Method for determining specific plasmid titers using RNA aptamers as molecular markers

Abstract

The present invention relates to an in vivo method for comparing the specific plasmid titers in two or more bacterial cell populations, using RNA aptamers.

Description

[0001] C012403-W001 / Gr

[0002] 1

[0003] METHOD FOR DETERMINING SPECIFIC PLASMIDE TITERS UNDER

[0004] USE OF RNA APTAMERS AS MOLECULAR MARKERS

[0005] The present invention relates to an in vivo method for comparing the specific plasmid titers in two or more bacterial cell populations using RNA aptamers, wherein the method comprises: a) providing one or more different plasmids, wherein the plasmid or the several different plasmids each comprise an expression cassette comprising a promoter functionally linked to a nucleic acid sequence and a terminator linked to the 3' end of the nucleic acid sequence, wherein the nucleic acid sequence exclusively encodes a molecule selected from the group of fluorogen-activating RNA aptamers; b) transforming each bacterial cell population with one plasmid from a); c) cultivating each bacterial cell population under defined cultivation conditions;d) Contacting each bacterial cell population with a binding partner for the fluorogen-activating RNA aptamer, whereby binding of the fluorogen-activating RNA aptamer expressed by the bacterial cells to the binding partner generates a fluorescence signal; and e) Detecting the fluorescence signal of the bacterial cells of the individual bacterial cell populations; f) Comparing the fluorescence signals of the bacterial cells of the individual bacterial cell populations, wherein a stronger normalized fluorescence signal indicates a higher specific plasmid titer in the respective bacterial cell population. C012403-W001 / Gr;

[0006] 2

[0007] The principle behind many new therapies is the use of genetic information for drug synthesis in a target cell. For this purpose, the genetic information is provided in the form of a target gene. To achieve efficient transfer of the target gene into a target cell, the target gene must first be provided in sufficient quantity. Plasmids or plasmid vectors represent flexible and easily modifiable sources of recombinant DNA for a wide variety of research, industrial, and pharmaceutical applications. Particularly in the field of modern medicine or gene therapy, plasmids serve, for example, as important starting materials in the production of mRNA or viral vectors, and can also be used themselves as active substances, for example, in the form of DNA vaccines.The development and application of new technologies has led to a sharp increase in the demand for high-quality plasmid DNA (pDNA) in recent years. Since the supply of large quantities of highly purified pDNA is often a limiting factor, optimizing existing pDNA production processes is of central importance.

[0008] Traditionally, pDNA is produced via fermentation using recombinant bacteria, such as Escherichia coli. For this purpose, a selected bacterial strain is transformed with a plasmid carrying a target gene and then cultured under suitable conditions. During cultivation and cell division, the bacterial cells replicate and multiply the contained plasmid and thus the target gene. This process is called cloning and enables efficient and sequence-accurate duplication of any target gene. The amount of plasmid, and therefore of the target gene, that can be obtained from each individual bacterial cell is determined by the frequency or efficiency of the C012403-W001 / Gr

[0009] 3

[0010] Plasmid replication is determined. The frequency and efficiency of plasmid replication, in turn, depend on factors such as the properties of the bacterial cell strain used for production, regulatory elements on the plasmid vector, the origin of replication of the plasmid backbone, the nutrients and additives present in the culture medium, and other cultivation parameters such as temperature and pH. The multitude of parameters influencing plasmid production results in a large number of possible combinations. Furthermore, the optimal value of individual parameters can depend on the target gene being cloned. Therefore, in addition to determining the best production conditions in general, these conditions must also be specifically optimized and adapted for each plasmid or target gene. To determine how the various parameters affect plasmid production, the average plasmid copy number (RPN) or...To determine the effect of the specific plasmid titer in bacterial cells, it is necessary to determine, taking all parameters into account, how many plasmid molecules are formed in the population of production cells or in each individual cell.

[0011] Various methods for determining the specific plasmid titer or quantifying the plasmid copy number are known in the prior art. Some methods for optimizing plasmid production involve an absolute quantification of the specific plasmid titer, while others involve a relative determination.

[0012] A well-known method for determining the specific plasmid titer involves isolating total bacterial DNA, which includes both chromosomal and pDNA, preparing a dilution series of the isolates, and performing gel electrophoretic analysis. C012403-W001 / Gr

[0013] Separation and subsequent visualization and analysis by ethidium bromide staining. By comparing the signals for chromosomal DNA and plasmid DNA, the plasmid copy number can be absolutely determined. However, electrophoretic separation of isolated DNA fractions is both time-consuming and error-prone, and can only be performed at low throughput (Pushnova et al. An easy and accurate agarose gel assay for quantitation of bacterial plasmid copy numbers. Anal Biochem. 2000 Aug 15; 284(1): 70-6).

[0014] Another method for determining specific plasmid titers is quantitative real-time PCR (qPCR) (Lee et al. Absolute and relative qPCR quantification of plasmid copy number in Escherichia coli. J Biotechnol. 2006 May 29; 123(3): 273-80). This PCR method uses specific primer pairs for regions on the bacterial chromosome as well as for regions on the plasmid. Intercalation of fluorescent dyes into the resulting amplicons generates a readable signal, enabling relative and absolute quantification of the pDNA. The specific primers required for performing qPCR must be provided or optimized in each case.While qPCR allows for a higher sample throughput than the previously described gel electrophoresis analysis, it requires additional procedural steps, such as pDNA linearization, as the presence of the compact supercoil structure can distort the results. Another factor that can influence the performance of qPCR analyses is the purity of the samples being analyzed. This problem is solved in digital PCR by very high dilution during sample preparation. The principle of digital PCR is that DNA-containing samples are separated into different compartments. The sample is diluted so much that each C012403-W001 / Gr.

[0015] 5

[0016] Each compartment contains, on average, less than a single copy of a DNA molecule. A PCR reaction is then performed in each of the prepared compartments. The products of the PCR reaction are detected using intercalating and fluorescent dyes, with a detectable signal only appearing in those compartments that initially contained a DNA molecule.

[0017] In another method described in the prior art for the in vivo determination of plasmid copy number or specific plasmid titers, two plasmids independently present in the cell are used (Shao et al. Single-cell measurement of plasmid copy number and promoter activity. Nat Commun. 2021 Mar 5; 12(1): 1475). A reporter plasmid encodes the fusion protein PhiF-RFP, and a target plasmid has a number of binding sites for PhiF-RFP. This combination results in localized foci after expression and binding of PhiF-RFP to the target plasmid. The dimensions of these foci can be determined by fluorescence microscopy, and their density can be used to calculate the plasmid copy number or the specific plasmid titer. This method allows for a very accurate detection of the plasmid titer, but requires extensive adaptations of the bacterial cells.Plasmids are very complex to perform and not suitable for high throughput.

[0018] Plasmids can be used not only for cloning target genes, but also to promote or mediate the formation of metabolites that can be produced in bacteria and then isolated from them, for example, amino acids or heterologous proteins. For this purpose, production cells are transformed with plasmids that induce the heterologous expression of proteins or biosynthesis genes of the corresponding C012403-W001 / Gr

[0019] Six anabolic or catabolic metabolic pathways are mediated. Due to the required constitutive or inducible expression of these genes during the production process, the presence of the corresponding plasmids increases the metabolic burden within the production cells. This can lead to daughter cells that, after cell division, lack a plasmid molecule due to an asymmetric distribution of the available plasmid molecules, having a growth advantage over daughter cells that still contain one or more plasmid molecules after cell division. Product formation is reduced or completely inhibited in cells lacking a plasmid molecule compared to cells containing one. For the production process, cells lacking a plasmid molecule are therefore considered contaminants, as they continue to consume nutrients but do not support product formation to the intended extent.To design efficient and resource-saving production processes, the persistence of plasmids in cells or cell populations, or the loss rate, must be determined. For the methods used in the prior art, either plasmid DNA must be isolated, or the production cells must be isolated and cultured on selective media. The isolation of plasmid molecules is labor-intensive and not suitable for high throughput.

[0020] Methods for determining the loss rate of plasmids include replica plating of bacteria from antibiotic-free nutrient plates to antibiotic-containing nutrient plates. By counting the colony-forming units on both culture media, it can then be determined what proportion of the population carries a resistance-conferring plasmid. However, this method is labor-intensive and time-consuming. Other methods for determining the loss rate of plasmids C012403-W001 / Gr

[0021] 7 are based on qPCR, which requires a complex and error-prone DNA isolation process.

[0022] Aptamers are short, synthetic RNA, DNA, or peptide molecules with a specific three-dimensional structure and the ability to bind with high affinity to a target molecule. The production and selection of aptamers is comprehensively described in the prior art and well known to those skilled in the art (e.g., EP3995575 Al).

[0023] The use of aptamers to visualize RNA molecules and small metabolites in living cells is known in the prior art. In this process, the RNA aptamers bind to small target molecules and induce their fluorescence. As long as no binding occurs, neither the aptamer nor the target molecule is fluorescent; only the binding of the aptamer to the target molecule activates the fluorescence (Filonov and Jaffrey. RNA Imaging with Dimeric Broccoli in Live Bacterial and Mammalian Cells. Curr Protoc Chem Biol. 2016 Mar 16; 8 (1): l-28).

[0024] One object of the present invention is to provide a method for determining specific plasmid titers in bacterial cell populations, which avoids one or more disadvantages of the methods described in the prior art and which can be carried out in vivo. Another object of the invention is a method for comparing the specific plasmid titers in two or more bacterial cell populations, which can be carried out in vivo.

[0025] For example, one object of the invention is to provide a simple method for determining or comparing specific plasmid titers, which enables a high throughput rate, and wherein a labor-intensive isolation of the C012403-W001 / Gr

[0026] 8

[0027] Plasmid DNA from the bacterial cells is not required. A further object of the invention is to provide a method for determining or comparing specific plasmid titers, which enables quantification of the plasmid copy number, both at the level of a bacterial cell population and at the level of individual cells. Another object of the invention is to provide a method for determining or comparing specific plasmid titers, wherein no complex modifications of the plasmids are required. A further object of the invention is to provide a method for determining or comparing specific plasmid titers, wherein the reporter system used does not significantly affect bacterial physiology, represents the lowest possible metabolic burden for the bacterial cell, and wherein the reporter system enables the most direct possible readout method.

[0028] Another object of the invention is to provide a method suitable for determining the influence of the plasmid backbone and / or regulatory elements located on the plasmid on the specific plasmid titer or copy number of a plasmid in bacterial cells. A further object of the invention is to provide a method suitable for determining the influence of cultivation conditions and / or the genotype of the bacterial strain used for plasmid production on the specific plasmid titer or copy number of a plasmid in bacterial cells. A further object of the invention is to provide a method for determining the loss rate of a plasmid in bacterial cells, which can be carried out in vitro. C012403-W001 / Gr

[0029] 9

[0030] The present invention solves these problems by an in vivo method for comparing the specific plasmid titers in two or more bacterial cell populations using RNA aptamers, wherein the method comprises: a) providing one or more different plasmids, wherein the plasmid or the several different plasmids each comprise an expression cassette comprising a promoter functionally linked to a nucleic acid sequence and a terminator linked to the 3' end of the nucleic acid sequence, wherein the nucleic acid sequence exclusively codes for a molecule selected from the group of fluorogen-activating RNA aptamers; b) transforming each bacterial cell population with one plasmid from a); c) cultivating each bacterial cell population under defined cultivation conditions;d) Contacting each bacterial cell population with a binding partner for the fluorogen-activating RNA aptamer, whereby binding of the fluorogen-activating RNA aptamer expressed by the bacterial cells with the binding partner generates a fluorescence signal; and e) Detecting the fluorescence signal of the bacterial cells of the individual bacterial cell populations; f) Comparing the fluorescence signals of the bacterial cells of the individual bacterial cell populations, whereby a stronger normalized fluorescence signal indicates a higher specific plasmid titer in the respective bacterial cell population.

[0031] The invention is based on the integration of DNA sequences leading to the expression of fluorogen-activating RNA aptamers onto plasmids. These fluorogen-activating RNA aptamers are expressed by the bacterial cells containing the plasmid, C012403-W001 / Gr

[0032] 10. After the bacterial cells are brought into contact with binding partners for the respective RNA aptamers, for example fluorogens, the fluorogen-activating RNA aptamers bind to the binding partners, generating a fluorescence signal. This fluorescence signal indicates the presence of the fluorogen-activating RNA aptamers and thus the plasmids encoding them. According to the invention, the presence of a plasmid can therefore be detected in vivo by a fluorescence signal.

[0033] According to the invention, the DNA sequences leading to the expression of fluorogen-activating RNA aptamers are provided in an expression cassette on the respective plasmids. According to the invention, all plasmids of the inventive method each comprise an identical expression cassette. As used herein, an identical expression cassette is an expression cassette in which the promoter, the nucleic acid sequence, and the terminator each have the same or substantially the same function or sequence as in the reference cassette, and in which the expression cassette has a homology of at least 90%, preferably at least 95%, particularly preferably at least 98% to the reference cassette.

[0034] The present invention provides a comparative method in which the expression cassette functions as the measuring unit. To obtain a valid comparison, this measuring unit, i.e., the expression cassette, must be kept constant within an experiment. For a valid comparison of the specific plasmid titers between two or more bacterial cell populations, it is therefore essential that the expression cassette used for generating the fluorescence signal is identical in all plasmids being compared. This prevents differences in the measured fluorescence signal caused by the reporter system.

[0035] 11 can be directly attributed to variations in the plasmid itself (e.g. in the plasmid backbone or in regulatory elements) or in the genotype of the bacterial cell populations.

[0036] The method according to the invention can alternatively be used to quantify other DNA molecules, in particular extrachromosomal DNA molecules, in the cell by linking the DNA molecules to an RNA aptamer reporter, in particular an expression cassette according to the invention. Examples of DNA molecules that can be used are, for example, chromosomal DNA, minicircle DNA, linear DNA, covalently closed DNA, covalently closed DNA fragments, doggybone DNA or single-stranded DNA.

[0037] Quantifying the fluorescence signal allows conclusions to be drawn about the specific plasmid titer or plasmid copy number. The fluorescence signal generated after the binding of a fluorogen-activating RNA aptamer and its binding partner is directly proportional to the number of RNA aptamer molecules present. If a bacterial cell contains few plasmid molecules, it also contains few sequences encoding RNA aptamers. Consequently, relatively few fluorogen-activating RNA aptamers are synthesized in such a cell, resulting in a relatively weak fluorescence signal. Conversely, if a cell contains many plasmid molecules, it also contains many sequences encoding RNA aptamers. Consequently, relatively many fluorogen-activating RNA aptamers are synthesized in such a cell, resulting in a relatively strong fluorescence signal.

[0038] According to the invention, it could be shown that bacterial cells exhibit a relatively weak fluorescence signal when they have been transformed with a plasmid which, for example, due to the present replicon, has a low copy number in the cell and consequently a low specific plasmid titer in the bacterial cell population, and exhibit a relatively strong fluorescence signal when they have been transformed with a plasmid which, for example, due to the present replicon, has a high copy number in the cell and consequently a high specific plasmid titer in the bacterial cell population.

[0039] The method according to the invention enables the determination of the specific plasmid titer in a bacterial cell population, or the comparison of specific plasmid titers in one or more bacterial cell populations. Here, a bacterial cell population with a specific plasmid copy number or a specific plasmid loss rate can be compared with one or more other bacterial cell populations in order to identify the bacterial cell population that exhibits the highest possible specific plasmid titer. A high specific plasmid titer in a bacterial cell population is caused by a high plasmid copy number in the individual bacterial cells of the population.The plasmid copy number, in turn, can be influenced by a wide variety of factors, such as the plasmid backbone, regulatory elements on the plasmid (e.g., replicons), promoters, target genes, selection markers, homopolymeric regions and / or repeat sequences, the genotype of the bacterial strain, the plasmid loss rate, and / or cultivation conditions.

[0040] A high specific plasmid titer in a bacterial cell population can occur, for example, if a proportion of the bacterial cells in the population have a high plasmid copy number, or if the plasmid loss rate in the population is low, i.e., if the proportion of plasmid-bearing cells is low.

[0041] 13

[0042] Bacterial cells are high. A lower plasmid titer in a bacterial cell population can be caused, for example, by a lower average plasmid copy number in the bacterial cells of the population, or by a high plasmid loss rate, if, for example, only 50% of the bacterial cells carry the plasmid.

[0043] For the economical production of plasmids, a high plasmid titer or a high specific plasmid titer is generally desired. According to the invention, a simple and rapid method is provided to investigate the influence of individual parameters or combinations of several of the aforementioned parameters on the specific plasmid titer, and thus to identify parameters or combinations of parameters that yield the highest possible specific plasmid titer for a particular plasmid, in a particular bacterial strain, under specific cultivation conditions, and / or for a specific target gene. This, in turn, enables the selection of bacterial populations, plasmids, and / or cultivation conditions that provide a high specific plasmid titer.

[0044] The method according to the invention can, for example, be used to determine in vitro the effect of an adaptation or modification of a plasmid backbone on the specific plasmid titer, for example, a change in the replicates of the plasmid or the introduction of one or more inserts into the plasmid backbone. The method according to the invention can also be used to determine the effect of an adaptation or modification of the genotype of a bacterial strain on the specific plasmid titer. The method according to the invention can also be used, for example, to determine the effect of an adaptation or modification of the cultivation conditions of a bacterial cell population on C012403-W001 / Gr

[0045] 14 the specific plasmid titer. Thus, the method according to the invention enables, for example, the selection of plasmids or plasmid variants, bacterial genotypes and / or cultivation conditions with which high specific plasmid titers can be achieved by determining and comparing the fluorescence emission.

[0046] Furthermore, the in vivo method according to the invention can be used to determine the proportion of a bacterial cell population in which a plasmid, for example a production plasmid, is present, and the proportion of the bacterial cell population in which the plasmid was lost during cultivation. Due to the simple and rapid feasibility of measuring fluorescence, particularly with flow cytometric analysis, informed decisions regarding the continuation of the production process can be made in the sense of in-process control.

[0047] RNA aptamers are small molecules with a length of less than approximately 100 base pairs. This makes them significantly smaller than the mRNA synthesized during the transcription of a reporter protein gene, such as GFP. Furthermore, when using RNA aptamers as reporter molecules, no subsequent translation is necessary, as a reporter molecule already exists at the RNA level. Therefore, neither amino acids nor activated tRNAs are consumed, nor is the pool of free ribosomes required for cell growth burdened. RNA aptamers as reporter molecules thus represent no, or only a negligible, metabolic burden for the bacterial cell.

[0048] Since only one processing step is required when using RNA aptamers to generate the reporter molecule, namely C012403-W001 / Gr

[0049] 15. Since the transcription of an expression cassette is necessary, using RNA aptamers as reporter molecules allows for a more accurate determination of the specific plasmid titer than using protein reporters, whose production requires two processing steps: transcription and translation. Furthermore, the use of fluorogen-activating RNA aptamers as reporter molecules for determining the specific plasmid titer enables high-throughput screening by fluorescence assay, for example, using FACS. Plasmid isolation is not required, and the detection of the fluorescence signal is possible in vivo without requiring bacterial cell lysis, using various readily available methods such as flow cytometry, fluorescence microscopy, fluorescence spectroscopy, or fluorometry.

[0050] Definitions

[0051] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as they are commonly understood by a person skilled in the field of the present invention. Each technical feature mentioned in the following definitions can be applied to any embodiment of the invention.

[0052] The term "nucleic acid" refers to a sequence of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. These terms include single-stranded and double-stranded DNA or RNA, genomic DNA, cDNA, mRNA, saRNA, gRNA, siRNA, miRNA, or circRNA, which are purine and pyrimidine bases, nucleotide analogues, or other naturally occurring molecules. C012403-W001 / Gr

[0053] It may include 16 occurring, chemically or biochemically modified, non-natural or derivatized nucleotide bases.

[0054] The terms “nucleic acid sequence”, “nucleotide sequence”, “DNA sequence” and “RNA sequence” are known to a specialist and refer to an individual and specific sequence of nucleotides.

[0055] Homologous sequences, as used here, are defined as DNA sequences that are 90%, preferably at least 95%, and most preferably at least 98% identical. The degree of DNA identity is determined by the program "nucleotide blast," found at http: / / blast.ncbi.nlm.nih.gov / , which is based on the blastn algorithm. Predefined parameters were used as algorithm parameters for aligning two or more nucleotide sequences. The predefined general parameters are: Max target sequences = 100; Short queries = "Automatically adjust parameters for short input sequences"; Expect Threshold = 10; Word size = 28; Automatically adjust parameters for short input sequences = 0. The corresponding predefined scoring parameters are: Match / Mismatch Scores = 1, -2; Gap Costs = Linear.

[0056] The terms "plasmid," "plasmid vector," "pDNA," and "plasmid DNA" are used interchangeably herein and describe a construct of extrachromosomal genetic material, preferably a circular DNA duplex molecule, that can replicate in a cell independently of chromosomal DNA. The term "plasmid" is used in its broadest sense and includes any vehicle for a nucleic acid or target gene that, for example, makes it possible to introduce the nucleic acid or target gene into prokaryotic and / or eukaryotic host cells and, if desired, to integrate it into a genome. C012403-W001 / Gr

[0057] Plasmids can be, for example, expression vectors, cloning vectors, transfer vectors, or storage vectors, or combinations of these. An expression vector can be used to produce expression products, such as mRNA, peptides, polypeptides, or proteins.

[0058] The term plasmid variant here refers to a variant of a plasmid. Plasmid variants preferably differ from a parent plasmid in one or more sequence segments or regulatory elements. For example, one or more nucleotides may be exchanged, deleted, or inserted. Such sequence segments or regulatory elements can be, for example, a replicate, a promoter, a target gene, a selection marker, a homopolymeric region, or a repeat sequence. Preferably, plasmid variants differ from a parent plasmid in only a single sequence segment or a defined regulatory element; all other sections of the plasmid are essentially the same as in the parent plasmid. Furthermore, preferably, the modification of a sequence segment or...No additional regulatory element was introduced into the plasmid backbone that influences the expression of the expression cassette.

[0059] The terms "origin of replication" and "replicas" are used interchangeably here and refer to a specific sequence at which DNA replication is initiated. The term origin of replication includes both prokaryotic, e.g., bacterial, origins of replication, and eukaryotic origins of replication, for example, from mammals. C012403-W001 / Gr

[0060] 18

[0061] The term "promoter" describes a DNA regulatory region located upstream (5') of a coding sequence of a gene or a non-coding sequence, capable of binding an RNA polymerase and triggering the transcription of a downstream (3') coding or non-coding sequence. Suitable promoters can be derived from any organism, including prokaryotic and eukaryotic organisms. A promoter can control the transcription of a prokaryotic or eukaryotic gene. A promoter may also include additional recognition or binding sites for other factors involved in the regulation of gene transcription. A promoter can be a constitutively active promoter, i.e., a promoter that is constitutively in an active state, or it can be an inducible promoter, i.e.A promoter is a promoter whose state is controlled by an external stimulus, for example, being switched from an inactive to an active state. Such an external stimulus could be, for example, a specific temperature, a compound, or a protein. Constitutively active and inducible promoters are known to those skilled in the art. Examples of inducible promoters include the lac promoter, which is inducible by isopropyl β-D-thiogalactopyranoside (IPTG), the tetracycline-regulated promoter, the rhamnose-inducible promoter, and the arabinose promoter, which is inducible by arabinose.

[0062] As used herein, “functionally associated with a promoter” means that the promoter causes or regulates the transcription of DNA that codes for a gene. The term “gene” here refers to a nucleic acid sequence that can be transcribed into functional RNA. The term “gene” is used here for both protein-coding and non-coding genes. C012403-W001 / Gr

[0063] The term "terminator sequence" describes a nucleic acid sequence that determines the end of a transcript, gene or operon, as it leads to the termination of transcription.

[0064] The term "plasma titer" describes the amount of

[0065] Plasmid product consisting of a defined volume of

[0066] A bacterial culture can be isolated. The plasmid titer is measured as...

[0067] Concentration of the plasmid product given in mg / L or nmol / L.

[0068] The term "specific plasmid titer" describes the amount of plasmid product that can be isolated from a defined volume of a bacterial culture relative to the number of bacterial cells in that culture. The specific plasmid titer is expressed as the concentration of plasmid product per unit of optical density in mg / L / ODeoo or nmol / L / ODeoo.

[0069] The term "plasmid copy number" describes the number of

[0070] Plasmids of the same sequence in a bacterial cell.

[0071] Insofar as a comparison of plasmids or bacterial cell populations is described herein, it is always stipulated that the expression cassette used for signal generation, comprising a promoter functionally linked to a nucleic acid sequence and a terminator linked to the 3' end of the nucleic acid sequence, is identical in its configuration in all plasmids to be compared; that is, the same promoter, the same terminator, and a nucleic acid sequence encoding the same molecule selected from the group of fluorogen-activating RNA aptamers are always used. This ensures the validity and comparability of the measured values.

[0072] Fluorescence signals are reliable. Furthermore, the invention provides for C012403-W001 / Gr

[0073] 20 that the same binding partner is always used for a specific fluorogen-activating RNA aptamer.

[0074] The terms “plasmid loss rate” and “plasmid persistence” are used interchangeably herein and describe the ability of a plasmid to persist in a microbial population for one or more generations under selective or non-selective conditions, and are expressed at a given time as the percentage of plasmid-bearing bacterial cells in a total population of bacterial cells.

[0075] As used herein, the term "bacterial cell population" means the totality of bacterial cells within a spatially confined culture. The individual bacterial cells can be either genetically identical or genetically different.

[0076] As used herein, “genotypically identical” bacteria or bacterial cell populations have identical or substantially identical genomic elements, i.e., an identical or substantially identical bacterial genome and identical or substantially identical extrachromosomal genetic elements, e.g., plasmids, wherein plasmids which have an expression cassette according to the invention are not extrachromosomal genetic elements within the meaning of the present invention.

[0077] The term "cultivation conditions" describes all biotic and abiotic environmental conditions of bacterial cells during growth and reproduction. These include, for example, temperature, pH value, oxygen content, nutrient content, stirring speed, and the presence or absence of antibiotics and other media additives. C012403-W001 / Gr

[0078] The term "aptamer" here refers to a single-stranded, partially single-stranded, partially double-stranded, or double-stranded nucleotide sequence with a specific binding affinity for selected target molecules or a group of molecules. In some cases, the aptamer recognizes the target molecule through a mechanism other than classical Watson-Crick base pairing.

[0079] The term "RNA aptamer," as used herein, describes an aptamer comprising ribonucleoside units, for example, a small RNA molecule with fewer than about 100 base pairs. Other preferred RNA aptamers comprise a sequence of direct, complementary, or inverted repeats of ribonucleoside units leading to the formation of one RNA aptamer molecule or multiple RNA aptamer units within one molecule. Particularly preferred are the direct, complementary, or inverted repeats of the ribonucleoside units leading to the formation of an RNA aptamer linked by ribonucleoside units that serve as stabilizing connecting elements.

[0080] The term “fluorogen-activating RNA aptamers”, as used herein, refers to highly structured RNA molecules which, due to their structure, can complex a specific binding partner, for example a low molecular weight fluorogen, and induce its fluorescence.

[0081] The term "fluorescence signal" here refers to light waves of a specific frequency range that emanate from the object under investigation and can be detected by optical methods. The frequency range can lie both within and outside the range of visible light. C012403-W001 / Gr

[0082] 22

[0083] From a detailed description of the invention

[0084] The present invention relates to an in vivo method for comparing the specific plasmid titers in two or more bacterial cell populations using RNA aptamers, wherein the method comprises: a) providing one or more different plasmids, wherein the plasmid or the several different plasmids each comprise an expression cassette comprising a promoter functionally linked to a nucleic acid sequence and a terminator linked to the 3' end of the nucleic acid sequence, wherein the nucleic acid sequence exclusively encodes a molecule selected from the group of fluorogen-activating RNA aptamers; b) transforming each bacterial cell population with one plasmid from a); c) cultivating each bacterial cell population under defined cultivation conditions;d) Contacting each bacterial cell population with a binding partner for the fluorogen-activating RNA aptamer, whereby binding of the fluorogen-activating RNA aptamer expressed by the bacterial cells with the binding partner generates a fluorescence signal; and e) Detecting the fluorescence signal of the bacterial cells of the individual bacterial cell populations; f) Comparing the fluorescence signals of the bacterial cells of the individual bacterial cell populations, whereby a stronger normalized fluorescence signal indicates a higher specific plasmid titer in the respective bacterial cell population.

[0085] According to the invention, RNA aptamers are used as molecular markers to determine specific plasmid titers. (C012403-W001 / Gr)

[0086] 23. According to the inventive method, the specific plasmid titer of any plasmid can be determined. For example, the specific plasmid titer of plasmids based on pUC18, pUC19, pBR322, pR6K, pSCl 01, or pACYC184 can be determined. However, the plasmids mentioned represent only a small selection of possible plasmids. Further plasmids are generally known to those skilled in the art and are also described in the relevant literature.

[0087] The present invention provides an in vivo method for comparing the specific plasmid titers in two or more bacterial cell populations.

[0088] According to the invention, the specific plasmid titer or plasmid copy number in a bacterial cell population under investigation can be determined by comparing the signal strength of a fluorescence signal from the bacterial cell population under investigation with the signal strength of a fluorescence signal from a reference value. For example, the fluorescence signal of a bacterial cell population or of bacterial cells without a plasmid can be used as a reference value, or the fluorescence signal of a bacterial cell population or of bacterial cells with a plasmid having a known plasmid copy number can be used. In one embodiment of the invention, the fluorescence signal of a bacterial cell population or of bacterial cells from one experimental setup is compared with that of another experimental setup from a different bacterial cell population, and the fluorescence signals of both experimental setups are evaluated relative to each other.

[0089] In a preferred embodiment of the invention, the

[0090] Plasmid whose specific titer was determined or compared C012403-W001 / Gr

[0091] 24. A selection marker is to be used, wherein the selection marker is preferably selected from the group consisting of an antibiotic resistance gene, a toxin, an antitoxin, and a reporter element. Furthermore, auxotrophy markers are suitable as selection markers, which encode an essential gene that is deleted in the respective bacterial strain containing the plasmid. The selection marker is suitable for distinguishing bacterial cells containing the plasmid from bacterial cells that do not contain the plasmid. Numerous selection markers are known in the field. Suitable genes that confer antibiotic resistance are known to a person skilled in the art.The preferred antibiotic is the one against which resistance is conferred by the selection marker, selected from the group consisting of ampicillin, tetracycline, kanamycin, geneticin, chloramphenicol, spectinomycin, hygromycin, sulfonamide, trimethoprim, bleomycin, zeocin, gentamicin, and blasticidin. Suitable selection markers also include toxins such as sacB and hok, antitoxins such as sacB antisense RNA and sok, and reporter elements such as GFP.

[0092] According to the invention, any bacterial strain suitable for plasmid production can be used. Suitable bacterial strains for plasmid production are well known to those skilled in the art. The bacterial strain is preferably a Gram-negative bacterium, more preferably a bacterial strain of the genus Enterobacteriaceae, particularly preferably a strain of the species Escherichia coli (E. coli), especially preferably E. coli K12 or E. coli B.

[0093] The two or more bacterial cell populations may have the same or a similar genotype or genetic background, or they may have two or more different genetic backgrounds or genotypes. C012403-W001 / Gr

[0094] 25. In one embodiment of the invention, the different genotypes can be generated by irradiating a starting strain. The two or more bacterial cell populations can originate from the same bacterial strain, or they can originate from two or more different bacterial strains.

[0095] According to the invention, in step a) a plasmid or several different plasmids are provided. Each of the plasmids comprises an expression cassette according to the invention. Preferably, each of the plasmids comprises exactly one expression cassette according to the invention.

[0096] The expression cassette according to the invention can be located at any suitable position and in any relative orientation within a plasmid. A person skilled in the art is able to identify suitable sites within a plasmid for the integration of the expression cassette. Preferably, the expression cassette is integrated such that neighboring sequence segments or elements do not influence the transcription of the expression cassette.

[0097] Preferably, all plasmids of a method according to the invention have a substantially same relative orientation of the expression cassette.

[0098] In a preferred embodiment, the expression cassette is inserted into a cloning site in the plasmid in question. The insertion or cloning of the expression cassette into a plasmid can be carried out by any method known in the field. Methods for cloning DNA sequences are well known to a person skilled in the art. C012403-W001 / Gr

[0099] 26

[0100] The plasmids used in the process according to the invention each comprise the same expression cassette. The expression cassette each comprises the same promoter, which is functionally linked to the same nucleic acid sequence, and each comprises the same terminator, which is linked to the 3' end of the nucleic acid sequence, wherein the nucleic acid sequence each encodes the same molecule selected from the group of fluorogen-activating RNA aptamers. According to the invention, the nucleic acid sequence encodes exclusively one molecule selected from the group of fluorogen-activating RNA aptamers, more preferably exclusively the same molecule selected from the group of fluorogen-activating RNA aptamers.

[0101] The expression cassette according to the invention comprises a promoter functionally linked to a nucleic acid sequence and a terminator linked to the 3' end of the nucleic acid sequence. Each expression cassette contains the same promoter, functionally linked to the same nucleic acid sequence. The respective promoter can be selected depending on the bacterial strain used, the plasmid used, the cultivation conditions used, and / or the RNA aptamer used. Any promoter suitable for expression in bacterial cells, particularly in the bacterial strain used, can be used. A multitude of suitable promoters for expression in bacterial cells are known to those skilled in the art. The promoter can be a constitutively active promoter, i.e.,a promoter that is constitutively in an active state, or it can be an inducible promoter, i.e., a promoter whose state is controlled by an external stimulus, for example by the inactive C012403-W001 / Gr.

[0102] 27 is brought into an active state. Such an external stimulus could be, for example, a specific temperature, a compound, or a protein. Constitutively active and inducible promoters are well known to a person skilled in the art.

[0103] The expression cassette according to the invention further comprises a nucleic acid sequence that exclusively codes for a molecule selected from the group of fluorogen-activating RNA aptamers. The expression cassette comprises a nucleic acid sequence, each sequence encoding the same molecule selected from the group of fluorogen-activating RNA aptamers. Fluorogen-activating RNA aptamers are small, highly structured RNA molecules which, due to their structure, can complex a specific binding partner, for example, a low-molecular-weight fluorogen, and induce its fluorescence. Numerous fluorogen-activating RNA aptamers and their specific binding partners are described in the prior art and are known to a person skilled in the art, for example, in EP2398775 B1; US ​​10444224 B2; US 11976382 B2 or US 9664676 B2.Likewise, methods for selecting RNA aptamers for binding to a molecule are widely known in the scientific community and readily accessible to a specialist.

[0104] The expression cassette according to the invention further comprises a terminator that is connected to the 3' end of the nucleic acid sequence encoding a molecule selected from the group of fluorogen-activating RNA aptamers. In the method according to the invention, the expression cassette comprises the same terminator that is connected to the 3' end of the nucleic acid sequence.

[0105] The terminator can be selected depending on the bacterial strain used, the plasmid C012403-W001 / Gr, the cultivation conditions, and / or the RNA aptamer. Any terminator suitable for transcriptional termination in bacterial cells, particularly in the specific bacterial strain used, can be employed. A person skilled in the art is aware of numerous suitable terminators.

[0106] In the expression cassette according to the invention, the promoter is functionally linked to the nucleic acid sequence, and the 3' end of the nucleic acid sequence is linked to a terminator sequence.

[0107] In a preferred embodiment, there is no ribosome binding site, no open reading frame, no 5' untranslated region, no 3' untranslated region, and no homopolymeric region located between the promoter and the nucleotide sequence.

[0108] In another preferred embodiment, there is no ribosome binding site, no open reading frame, no 5' untranslated region, no 3' untranslated region, and no homopolymeric region located between the nucleotide sequence and the terminator sequence.

[0109] In a particularly preferred embodiment, there is no ribosome binding site, no open reading frame, no 5' untranslated region, no 3' untranslated region, and no homopolymeric region located between the promoter and the nucleotide sequence, nor between the nucleotide sequence and the terminator sequence. In this embodiment, the fluorogen-activating RNA aptamer is not linked to any other coding or non-coding RNA. In this preferred embodiment, the nucleic acid sequence C012403-W001 / Gr encodes

[0110] 29 exclusively for the molecule selected from the group of fluorogen-activating RNA aptamers, and not for any other molecule or for a fusion molecule consisting of a fluorogen-activating RNA aptamer and any other molecule, or for a protein.

[0111] Preferred fluorogen-activating RNA aptamers for use in the present invention are, for example, Spinach Aptamer (Spinach), Spinach2 Aptamer (Spinach 2), F30-2xdBroccoli Aptamer, Broccoli Aptamer (Broccoli), Red Broccoli Aptamer (Red Broccoli), Orange Broccoli Aptamer (Orange Broccoli), Corn Aptamer (Maize), Beetroot Aptamer (Beetroot), Mango, Mango II, Mango III, Mango IV, Malachite Green Aptamer (Malachite Green Aptamer) and derivatives thereof. Particularly preferred fluorogen-activating RNA aptamers for use in the present invention are, for example, Spinach2 aptamer (Spinach2), Broccoli aptamer (Broccoli) and F30-2xdBroccoli aptamer.

[0112] According to the invention, in step b), each bacterial cell population in which the specific titer is to be determined or compared is transformed with a plasmid comprising the expression cassette. The transformation of the bacterial cell population can be carried out by any method known in the field. Methods for transforming bacterial cells are well known to those skilled in the art. Preferably, within the method according to the invention, the transformation of the bacterial cells is carried out for all bacterial cell populations using the same methods and under the same conditions.

[0113] In one embodiment of the invention, all

[0114] Bacterial cell populations are transformed with the same plasmid. In another formulation, the C012403-W001 / Gr

[0115] 30

[0116] Bacterial cell populations are each transformed with different plasmids, the different plasmids preferably comprising the same expression cassette. In another embodiment, two or more bacterial cell populations are transformed with the same plasmid, and one or more other bacterial cell populations with a different plasmid, the different plasmids preferably comprising the same expression cassette. In one experimental setup, for example, several bacterial cell populations can be transformed with plasmid A. In another experimental setup, for example, one bacterial cell population can be transformed with plasmid A, one bacterial cell population with plasmid B, and one bacterial cell population with plasmid C, the plasmids A, B, and C preferably comprising the same expression cassette.In another experimental approach, for example, two bacterial cell populations can be transformed with plasmid A, one or more bacterial cell populations with plasmid B, one or more bacterial cell populations with plasmid C, wherein plasmids A, B and C preferably comprise the same expression cassette.

[0117] In the inventive method, the two or more bacterial cell populations are cultured in one step (c). During cultivation, the fluorogen-activating RNA aptamer is expressed in the bacterial cells of the bacterial cell populations containing one or more copies of a plasmid. Since, according to the invention, the plasmids used comprise the same expression cassette, the same fluorogen-activating RNA aptamer is expressed in each case. Thus, during cultivation, heterologous expression of the fluorogen-activating RNA aptamers occurs in plasmid-bearing bacterial cells. C012403-W001 / Gr

[0118] 31

[0119] Step c) of cultivating the bacterial cell is carried out for each bacterial cell population under defined cultivation conditions. In one embodiment of the invention, the cultivation conditions for two or more bacterial cell populations are essentially the same. In another embodiment, the cultivation conditions for each of the bacterial populations used are different.

[0120] In another implementation, where more than two bacterial cell populations are investigated, essentially identical cultivation conditions can be used for two or more bacterial cell populations, while different cultivation conditions can be used for one or more bacterial cell populations. For example, in one experimental setup, all bacterial cell populations can be cultivated under cultivation conditions A. In another experimental setup, for example, one bacterial cell population can be cultivated under cultivation conditions A, one bacterial cell population under cultivation conditions B, and one bacterial cell population under cultivation conditions C.In another experimental approach, for example, two bacterial cell populations can be cultivated under cultivation conditions A, one or more bacterial cell populations under cultivation conditions B, and one or more bacterial cell populations under cultivation conditions C.

[0121] Various parameters, such as nutrient supply, oxygen partial pressure, pH, stirring speed, and culture temperature, can be adjusted and controlled during the cultivation of bacterial cell populations. Suitable values ​​for these parameters for bacterial cultivation are well known to a person skilled in the art. In a preferred C012403-W001 / Gr

[0122] 32

[0123] In this form, cultivation takes place at a temperature between about 30 °C and about 42 °C, particularly preferably at about 37 °C.

[0124] Step c) of cultivating the bacterial cell populations can be carried out, for example, in a shake flask or a fermenter. In a preferred embodiment, cultivation takes place in a shake flask. In another preferred embodiment, cultivation takes place in a bioreactor. Media for cultivating bacteria in shake flasks and fermenters are known to those skilled in the art of microbial cultivation. They typically consist of a carbon source, a nitrogen source, and additives such as vitamins, salts, and trace elements, which optimize cell growth and plasmid production. All common media known to those skilled in the art for cultivating microorganisms are suitable as fermentation media. Complex media, mineral salt media, or minimal salt media to which a specific proportion of complex components, such as yeast extract, is added, can be used.Furthermore, additional components can be added to the medium to improve cell growth, such as vitamins, salts, amino acids, and / or trace elements. In a preferred embodiment, a complete medium is used. In a particularly preferred embodiment, an LB medium is used.

[0125] The cultivation of the individual bacterial cell populations is carried out, for example, over a period of approximately 1 to approximately 72 hours. In a preferred embodiment, cultivation is carried out over a period of approximately 8 to approximately 48 hours. In a further preferred embodiment, cultivation is carried out over a period of approximately 8 to approximately 24 hours. In a particularly preferred embodiment, cultivation is carried out using a C012403-W001 / Gr

[0126] 33

[0127] Cultivation takes place over several consecutive days, for example, 1 to 20 days, preferably up to 20 days, more preferably 10 to 14 days, and particularly preferably 14 days, for periods of approximately 8 to 48 hours each day. In yet another preferred embodiment, cultivation takes place over several consecutive days for periods of approximately 8 to 24 hours each day. In a further particularly preferred embodiment, cultivation takes place over several consecutive days for periods of approximately 8 to 12 hours each day. Preferably, the individual bacterial cell populations are cultivated for the same period in each case.

[0128] According to the invention, in step d), the individual bacterial cell populations are each brought into contact with a binding partner for the fluorogen-activating RNA aptamer. Since the individual bacterial cell populations preferably comprise the same expression cassette and thus express the same fluorogen-activating RNA aptamer, the individual bacterial cell populations are each brought into contact with the same binding partner for the fluorogen-activating RNA aptamer expressed in the process according to the invention. The individual bacterial cell populations are each brought into contact with the binding partner separately. This contacting can be carried out by any suitable method, for example, by titration, weighing, or mixing. The binding partner can, for example, be added to the culture medium. Preferably, the contacting is carried out by the same method.In a preferred embodiment, the binding partner and the individual bacterial populations are each converted into phosphate-buffered saline (PBS). C012403-W001 / Gr.

[0129] 34

[0130] The concentration of the binding partner in the culture medium or in the BBS is, for example, between about 1 and about 100 pM, preferably between about 20 and about 60 pM, and particularly preferably at about 40 pM. Preferably, the concentration of the binding partner in the culture medium is the same for all bacterial cell populations.

[0131] The two or more bacterial cell populations can be brought into contact with the same binding partner or with different binding partners. Preferably, the two or more bacterial cell populations are brought into contact with the same binding partner. Binding of the fluorogen-activating RNA aptamer expressed by the bacterial cells to the binding partner generates a fluorescence signal in the bacterial cell populations.

[0132] Binding partners for fluorogen-activating RNA aptamers are well known in the field. In a preferred embodiment of the invention, the binding partners for fluorogen-activating RNA aptamers are low-molecular-weight fluorogens. In another preferred embodiment, the binding partners are permeable to bacterial cells and passively diffuse across the bacterial membrane and cell wall into the bacterial cytoplasm. This means that the bacterial cells or the bacterial membrane are permeable to the binding partners.

[0133] Preferred binding partners for fluorogen-activating RNA aptamers are selected from the group consisting of DFHBI, DFHBI-1T, DFHO, DMHBI, and TOl-biotin; a particularly preferred binding partner is DFHBI-1T.

[0134] Preferred combinations of fluorogen-activating RNA aptamers and binding partners are selected from group C012403-W001 / Gr

[0135] 35 consisting of Spinach Aptamer (Spinach) and DFHBI, Spinach Aptamer (Spinach) and DFHBI-1T, Spinach2 Aptamer (Spinach2) and DFHBI, Spinach2 Aptamer (Spinach2) and DFHBI-1T, F30-2xdBroccoli Aptamer and DFHBI, F30-2xdBroccoli Aptamer and DFHBI-1T, Broccoli Aptamer (Broccoli) and DFHBI, Broccoli Aptamer (Broccoli) and DFHBI-1T, Mango and TOI-Biotin, Red Broccoli Aptamer (Red Broccoli) and DFHO, Red Broccoli Aptamer (Red Broccoli) and DFHBI, Red Broccoli Aptamer (Red Broccoli) and DFHBI-1T, Orange Broccoli aptamer (orange broccoli) and DFHO, orange broccoli aptamer (orange broccoli) and DFHBI, orange broccoli aptamer (orange broccoli) and DFHBI-1T. Particularly favored combinations of fluorogen-activating RNA aptamers and binding partners are broccoli aptamer (broccoli) and DFHBI-1T, as well as F30-2xdBroccoli aptamer and DFHBI-1T.

[0136] According to the invention, a fluorescence signal is generated by binding the fluorogen-activating RNA aptamer expressed by the bacterial cells to the binding partner and excitation with light of a specific wavelength.

[0137] In the method of the invention, in step e) the fluorescence signal of the bacterial cells of the individual bacterial cell populations is detected. The detection of the fluorescence signal can be carried out by any suitable method known in the field. For example, the fluorescence signal can be detected by fluorescence microscopy, fluorescence spectroscopy, or fluorometry.

[0138] In a preferred embodiment, wherein the binding partner is a low-molecular-weight fluorogen, an interaction occurs after binding of the fluorogen-activating RNA aptamer with the binding partner, resulting in an increase or change in fluorescence. C012403-W001 / Gr

[0139] 36

[0140] The possibilities for detecting fluorescence intensity or fluorescence emission are diverse; this can be achieved, for example, by flow cytometric analysis (FACS) at the single-cell level, by analysis in plate readers at the population level in liquid culture or agar plate culture, or by analysis with camera systems that allow the excitation and detection of fluorescence emission. Due to the flexible and simple detection method, the determination of the plasmid copy number can be carried out in high throughput according to the invention.

[0141] The detection and quantification of the fluorescence signal is preferably carried out using the same detection system and according to the same protocol.

[0142] According to the invention, the detection of the fluorescence signal is carried out in vitro; lysis of the bacterial cells is not required.

[0143] In one embodiment of the invention, the method according to the invention further comprises step el) normalizing the fluorescence signal. In the case that the cell number or cell density of the bacterial cell populations differs significantly from one another, normalization can achieve improved comparability of the fluorescence signals of the bacterial cells of the individual bacterial cell populations.

[0144] According to the invention, the normalization of the fluorescence signals is preferably carried out with regard to the biomass in the bacterial cell population, and particularly preferably with regard to the number of bacterial cells in the bacterial cell population. Suitable methods for determining the biomass and the number of bacterial cells in a bacterial cell population are known to those skilled in the art and are described, for example, by Beal et al. C012403-W001 / Gr

[0145] 37

[0146] (Beal et al. Robust estimation of bacterial cell count from optical density; Communications Biology (2020) 3: 512). Preferably, the number of bacterial cells in the bacterial cell population is determined by measuring the optical density at 600 nm. Alternatively, the number of bacterial cells in the bacterial cell population is determined by determining the biomass, for example, the wet biomass or the dry biomass. Preferably, the number of bacterial cells in the bacterial cell population is determined by determining the dry biomass. Particularly preferably, the number of bacterial cells in the bacterial cell culture is determined by flow cytometric analysis. In another particularly preferred embodiment, the number of bacterial cells in the bacterial culture is determined by microscopy.In another particularly preferred embodiment, the number of bacterial cells in the bacterial cell population is determined by counting colony-forming units after serial dilution and plating on agar plates.

[0147] The fluorescence signal can be normalized, for example, by dividing the measured fluorescence intensity of the bacterial cell population by the optical density at 600 nm of the respective bacterial cell population, by dividing the measured fluorescence intensity of the bacterial cell population by the biomass in the respective bacterial cell population, or by dividing the measured fluorescence intensity of the bacterial cell population by the number of bacterial cells in the respective bacterial cell population. Alternatively, the fluorescence signal can be normalized by calculating the average of the fluorescence intensities of all or individual events recorded for the respective bacterial cell population. Normalization is performed for all C012403-W001 / Gr

[0148] Fluorescence signals of the bacterial cells of the individual bacterial cell populations in a series of experiments in the same way.

[0149] In step f) of the inventive method, the fluorescence signals of the bacterial cells of the individual bacterial cell populations are compared, wherein a stronger normalized fluorescence signal indicates a higher specific plasmid titer in the relevant bacterial cell population.

[0150] In the inventive method, if the fluorescence signals of the bacterial cells of the individual bacterial cell populations are normalized, the normalized fluorescence signals are compared, with a stronger normalized fluorescence signal indicating a higher specific plasmid titer in the relevant bacterial cell population.

[0151] In one embodiment, the in vitro method according to the invention further comprises step g) selecting the bacterial cell populations that exhibit the strongest fluorescence signal. This embodiment provides a method for screening for bacterial cell populations with a higher specific plasmid titer.

[0152] The method according to the invention allows, in one embodiment, for example in vitro, the effect of an adaptation or modification of a plasmid backbone on the specific plasmid titer to be determined. In one embodiment, a specific region of the plasmid, for example the origin of replication of the plasmid, is modified, or another specific sequence of a plasmid is modified, or one or more C012403-W001 / Gr

[0153] 39

[0154] Inserts are introduced into the plasmid backbone of a plasmid, and the specific plasmid titer of this modified plasmid is compared with the specific plasmid titer of the original plasmid. The position of the expression cassette is preferably not changed. In this configuration, the plasmids each contain the same expression cassette. In this configuration, the bacterial cell populations are essentially genotypically identical. In this configuration, the individual bacterial cell populations are cultivated under essentially identical cultivation conditions.

[0155] The method according to the invention can, in one embodiment, for example in vitro, determine the effect of an adaptation or change in the genotype or genome of a bacterial strain on the specific plasmid titer. For example, a starting bacterial strain can be irradiated, thereby providing genotypic variability. Preferably, in this embodiment, the specific plasmid titer in the bacterial cell population of the starting bacterial strain is compared with the specific plasmid titer in the resulting, genotypically modified bacterial cell populations.For example, if genotypically different bacterial cell populations are to be investigated using the method according to the invention, whereby specific coding sequence segments or regulatory elements such as promoters, terminators, ribosome binding sites, translation initiation regions or non-coding RNAs are to be investigated, preferably only the sequence segments or regulatory elements to be investigated are different; otherwise, the genotype is preferably the same or substantially the same. C012403-W001 / Gr.

[0156] 40

[0157] In this form of implementation, the plasmids each comprise the same expression cassette; preferably, the bacterial cell populations each comprise the same plasmid.

[0158] In this form of implementation, the cultivation of the individual bacterial cell populations takes place under essentially the same cultivation conditions.

[0159] The method according to the invention allows, in one embodiment, for example in vitro, the effect of an adjustment or change in the cultivation conditions of a bacterial cell population on the specific plasmid titer to be determined. In this embodiment, the plasmids each comprise the same expression cassette; preferably, the bacterial cell populations each comprise the same plasmid. In this embodiment, the bacterial cell populations are genotypically essentially identical.

[0160] The method according to the invention also allows the influence of combinations of the aforementioned parameters, i.e., the plasmid design, the cultivation conditions, and / or the bacterial genotype, to be determined. According to the invention, a method is provided in which, by determining and comparing fluorescence signals, a selection of plasmid variants, bacterial genotypes, and / or cultivation conditions can be carried out with which high specific plasmid titers can be achieved.

[0161] A first embodiment of the invention relates to an in vivo method for comparing the specific plasmid titers in two or more bacterial cell populations, wherein the individual bacterial cell populations are each transformed with a different plasmid. In this embodiment, C012403-W001 / Gr comprises

[0162] 41. The plasmids each use the same expression cassette. In this configuration, the bacterial cell populations to be transformed are essentially genotypically identical. In this configuration, the individual bacterial cell populations are cultivated under essentially identical cultivation conditions.

[0163] In this embodiment of the method according to the invention, the influence of the plasmid backbone, or the sequences contained on the plasmid, on the specific plasmid titer can be investigated. In this embodiment, the specific plasmid titer of two or more different plasmids or variants of a plasmid is compared in bacterial cell populations. The different plasmids can, for example, be plasmid variants. Plasmid variants have the same or a similar plasmid backbone. Furthermore, plasmid variants preferably include a selection marker, more preferably the same selection marker.

[0164] In the first embodiment of the invention, identical or different bacterial strains or bacterial cell populations, or bacterial strains or bacterial cell populations with different genotypes, can be used. Preferably, in this embodiment, the transformation of the plasmids into identical bacterial strains or bacterial cell populations with the same or substantially the same genotype is carried out.

[0165] In this embodiment of the inventive method, the transformation of the bacterial cell populations with the different plasmids is preferably carried out using the same method. C012403-W001 / Gr

[0166] 42

[0167] Cultivating each individual bacterial cell population under defined cultivation conditions can, in the first embodiment, be carried out for each bacterial cell population under different cultivation conditions, or for at least two of the bacterial cell populations under the same or substantially the same cultivation conditions, or for all bacterial cell populations under the same or substantially the same cultivation conditions. Preferably, the bacterial cell populations are cultivated under the same or substantially the same cultivation conditions. Furthermore, preferably, the bacterial cell populations are cultivated for the same or substantially the same cultivation duration.

[0168] Cultivation of the individual bacterial cell populations can be carried out under non-selective conditions, i.e. in a non-selective medium without selection markers, or in a selective medium, i.e. in a medium with selection markers.

[0169] The plasmids in the first embodiment preferably comprise a selection marker, more preferably all plasmids used comprise the same selection marker, and the cultivation of the individual bacterial cell populations preferably takes place in a selective medium in which plasmid-bearing bacterial cells have a growth advantage over non-plasmid-bearing bacterial cells.

[0170] In the first embodiment, the detection of the fluorescence signal can be carried out by any suitable method known in the field, whereby the fluorescence signal of the bacterial cells of the individual bacterial cell populations is always measured using the same method. Preferably, this is done using C012403-W001 / Gr

[0171] 43

[0172] Detection of the fluorescence signal, i.e., the fluorescence intensity or fluorescence emission of the bacterial cells of the individual bacterial cell populations at the population level in liquid culture by means of analysis in plate readers.

[0173] If normalization of the fluorescence signal is performed in the first embodiment, this can be done using any suitable method known in the field, for example, by dividing the determined fluorescence intensity of the bacterial cell population by the optical density at 600 nm of the respective bacterial cell population, or based on the total cell count or the number of viable cells. The normalization of the fluorescence signal is always performed using the same method. Preferably, the normalization of the fluorescence signal is performed by dividing the determined fluorescence intensity of the bacterial cell population by the optical density at 600 nm of the respective bacterial cell population.

[0174] A second embodiment of the invention relates to an in vivo method for comparing the specific plasmid titers in two or more bacterial cell populations, wherein the two or more different bacterial cell populations are genotypically distinct. In this embodiment, the plasmids each comprise the same expression cassette; preferably, the bacterial cell populations each comprise the same plasmid. In this embodiment, the individual bacterial cell populations are cultivated under substantially identical cultivation conditions.

[0175] In this form, the influence of the genetic background, for example the bacterial genotype of a bacterial strain, on the specific plasmid titer can be investigated. C012403-W001 / Gr

[0176] 44

[0177] In the second implementation, the bacterial cell populations can be transformed with the same plasmid, with different plasmids, or each with a different plasmid. Preferably, at least two of the bacterial cell cultures are transformed with the same plasmid. Even more preferably, all bacterial cell cultures are transformed with the same plasmid.

[0178] In this embodiment of the inventive method, the transformation of the two or more bacterial cell populations is preferably carried out using the same method.

[0179] In the second embodiment of the invention, different bacterial strains or bacterial cell populations, or bacterial strains or bacterial cell populations with different genotypes, are preferably used. Two or more different bacterial strains or bacterial cell populations with different genotypes can be used.

[0180] Cultivating the individual bacterial cell populations under defined cultivation conditions can, in the second embodiment, be carried out for each bacterial cell population under different cultivation conditions, or for at least two of the bacterial cell populations under the same or substantially the same cultivation conditions, or for all bacterial cell populations under the same or substantially the same cultivation conditions. Preferably, in this embodiment, the bacterial cell populations are cultivated under the same or substantially the same cultivation conditions for all bacterial cell populations. Furthermore, preferably, the cultivation of C012403-W001 / Gr

[0181] 45

[0182] Bacterial cell populations for the same or substantially the same cultivation duration.

[0183] Cultivation of the individual bacterial cell populations can be carried out under non-selective conditions, i.e. in a non-selective medium without selection markers, or in a selective medium, i.e. in a medium with selection markers.

[0184] The plasmid or the several different plasmids in the second embodiment preferably comprise a selection marker, particularly preferably all plasmids used comprise the same selection marker, and the cultivation of the individual bacterial cell populations preferably takes place in a selective medium in which plasmid-bearing bacterial cells have a growth advantage over non-plasmid-bearing bacterial cells.

[0185] In the second embodiment, the fluorescence signal can be detected using any suitable method known in the field, whereby the fluorescence signal of the bacterial cells of the individual bacterial cell populations is always measured using the same method. Preferably, the detection of the fluorescence signal, i.e., the fluorescence intensity or the fluorescence emission of the bacterial cells of the individual bacterial cell populations, is carried out at the population level in liquid culture by means of analysis in plate readers.

[0186] If normalization of the fluorescence signal is performed in the second implementation, this can be done using any suitable method known in the field, for example by dividing the determined fluorescence intensity of the bacterial cell population by the C012403-W001 / Gr

[0187] 46. ​​Optical density at 600 nm of the respective bacterial cell population or based on total cell count or viable cell count. The fluorescence signal is always normalized using the same method. Preferably, the fluorescence signal is normalized by dividing the determined fluorescence intensity of the bacterial cells of the bacterial cell population by the optical density at 600 nm of the respective bacterial cell population.

[0188] In a third embodiment, the invention relates to an in vivo method for comparing the specific plasmid titers in two or more bacterial cell populations using RNA aptamers, wherein the cultivation of the individual bacterial cell populations in step c) is carried out under different cultivation conditions. In this embodiment, the plasmids each comprise the same expression cassette; preferably, the bacterial cell populations each comprise the same plasmid. In this embodiment, the bacterial cell populations are genotypically essentially identical.

[0189] This third method allows for an investigation of the influence of cultivation conditions on the specific plasmid titer of a plasmid.

[0190] In the third embodiment of the invention, identical or different bacterial strains or bacterial cell populations, or bacterial strains or bacterial cell populations with different genotypes, can be used. Preferably, in this embodiment, the transformation of the plasmids into identical bacterial strains or bacterial cell populations with the same or substantially the same genotype is carried out.

[0191] In this form, the bacterial cell populations can be modified with the same plasmid or with different plasmids C012403-W001 / Gr

[0192] 47 or each be transformed with a different plasmid. Preferably, at least two of the bacterial cell cultures are transformed with the same plasmid; more preferably, all bacterial cell cultures are transformed with the same plasmid.

[0193] In the third embodiment of the method according to the invention, the transformation of the two or more bacterial cell populations is preferably carried out using the same method.

[0194] Preferably, the two or more bacterial cell populations are cultivated in this embodiment under cultivation conditions that differ in one or more parameters for at least two of the bacterial cell populations. Preferably, the cultivation conditions differ in one or more parameters for all bacterial cell populations. More preferably, the cultivation conditions differ in exactly one parameter for all bacterial cell populations. For example, the cultivation conditions differ with respect to temperature, pH, oxygen content, nutrient content, and / or stirring speed. Furthermore preferably, the bacterial cell populations are cultivated for the same or substantially the same cultivation duration.

[0195] Cultivation of the individual bacterial cell populations can be carried out under non-selective conditions, i.e., in a non-selective medium without selection markers, or in a selective medium, i.e., in a medium with selection markers. C012403-W001 / Gr

[0196] 48

[0197] The plasmid or the several different plasmids in the third embodiment preferably comprise a selection marker, particularly preferably all plasmids used comprise the same selection marker, and the cultivation of the individual bacterial cell populations preferably takes place in a selective medium in which plasmid-bearing bacterial cells have a growth advantage over non-plasmid-bearing bacterial cells.

[0198] In the third embodiment, the fluorescence signal can be detected using any suitable method known in the field, whereby the fluorescence signal of the bacterial cells of the individual bacterial cell populations is always measured using the same method. Preferably, the detection of the fluorescence signal, i.e., the fluorescence intensity or the fluorescence emission of the bacterial cells of the bacterial cell populations, is carried out at the population level in liquid culture by means of analysis in plate readers.

[0199] If, in the third embodiment, normalization of the fluorescence signal is performed, this can be carried out by any suitable method known in the field, for example, by dividing the determined fluorescence intensity of the bacterial cell population by the optical density at 600 nm of the respective bacterial cell population, or based on the total cell count or the number of viable cells. The normalization of the fluorescence signal is always performed using the same method. Preferably, the normalization of the fluorescence signal is performed by dividing the determined fluorescence intensity of the bacterial cells of the bacterial cell population by the optical density at 600 nm of the respective bacterial cell population. C012403-W001 / Gr

[0200] 49

[0201] The invention will be described in more detail below with reference to exemplary embodiments and the accompanying illustrations, without being limited by this.

[0202] Figure 1 shows a schematic plasmid map of the pVAXl plasmid. pCMV refers to the cytomegalovirus promoter. Kanamycin refers to the kanamycin resistance gene. BGH pA refers to the polyA region of the bovine growth hormone gene.

[0203] Figure 2 shows the fluorescence emission of E. coli bacterial colonies. Plasmid-bearing bacterial cells were detected using the Alexa 488 application in a ChemiDoc MP Imaging System (Bio-Rad). (A) E. coli WCM105 EfliC EyddS EendA after transformation with the reporter plasmid pVAXl F30-2xdBroccoli, cultured on LB agar with DFHBI-1T. Fluorescence emission is detectable in this bacterial cell colony. (B) Untransformed E. coli WCM105 EfliC EyddS EendA bacterial colonies, cultured on LB agar with DFHBI-1T. (C) E. coli WCM105 EfliC EyddS EendA bacterial colonies transformed with the control plasmid pVAXl and cultured on LB agar with DFHBI-1T. The bacterial cell colonies in both reactions (B) and (C) show no detectable fluorescence emission.

[0204] Figure 3 shows the determination of the fluorescence emission of bacterial cell populations in a Cytoflex SRT Gell Counter (Beckmann Coulter) at different time points. E. coli WCM105 EfliC EyddS EendA (light gray). E. coli WCM105 EfliC EyddS EendA, transformed with pVAXl F30-2xdBroccoli after incubation under selective conditions (dark gray; pVAXlF30-2dxBroccoli / Kan+). E. coli WCM105 EfliC EyddS EendA, transformed with pVAXl F30-2xdBroccoli under non-selective cultivation conditions (black; pVAXlF30-2dxBroccoli / Kan~). C012403-W001 / Gr

[0205] 50

[0206] Figure 4 shows the determination of fluorescence emission in a Cytoflex SRT Gell counter (Beckmann Coulter). E. coli WCM105 EfliC EyddS EendA, transformed with pVAXl (light gray; pVAXl). E. coli WCM105 EfliC EyddS EendA, transformed with pVAXl F30-2xdBroccoli (dark gray; pVAXl F30-2xdBroccoli). E. coli WCM105 EfliC EyddS EendA, transformed with pVAXl T112C F30-2xdBroccoli (black; pVAXl T112C F30-2xdBroccoli).

[0207] Figure 5 shows the mean fluorescence emission of E. coli WCM105 EfliC EyddS EendA bacterial cell populations transformed with pVAXl, pVAXl F30-2xdBroccoli or pVAXl T112C F30- 2xdBroccoli, as determined using the statistics function of the CytExpert SRT software.

[0208] Figure 6 shows the specific plasmid titer [mg / L / ODeoo] of the plasmids pVAXl T112C F30-2xdBroccoli and pVAXl F30-2xdBroccoli in shake flask culture.

[0209] Figure 7 shows the mean fluorescence emission of E. coli WCM105 EfliC EyddS EendA bacterial cell populations transformed with pVAXl, pVAXl F30-2xdBroccoli or pVAXl T112C F30-2xdBroccoli, as determined using a Biotek Synergy Hl plate reader (Agilent).

[0210] Figure 8 shows the specific plasmid titer of the plasmid variants 5bLl and 6bRl, which were selected according to the inventive method by screening for increased fluorescence emission, relative to the specific plasmid titer of the starting variant pVAXl T112C F30-2xdBroccoli. C012403-W001 / Gr

[0211] Examples

[0212] All enzymes and kits were used according to the manufacturers' instructions. Unless otherwise stated, all standard methods, molecular biological and microbiological procedures used, such as bacterial cell transformation, polymerase chain reaction (PCR), gene synthesis, DNA isolation and purification, DNA modification using restriction enzymes, Klenow fragment and / or ligase, were carried out in the manner described in the literature, recommended by the respective manufacturers, or in a manner well known to those skilled in the art.

[0213] Example 1. Production of an aptamer reporter plasmid ,

[0214] Transformation of bacteria and identification of

[0215] Transformant en

[0216] The plasmid pVAXl (Thermo Fisher Scientific, Figure 1) was used as the starting vector for the production of an aptamer reporter plasmid. pVAXl was linearized by cutting with the restriction enzymes BamHI and EcoRI (Thermo Fisher Scientific). A DNA fragment containing the aptamer variant F30-2xdBroccoli (SEQ ID NO:1) was generated by gene synthesis (GeneArt). Using this DNA fragment as a template and the oligonucleotides Pp576 (SEQ ID NO:2) and Pp444 (SEQ ID NO:3), the 332 base pair amplicon PCR01 (SEQ ID NO:4) containing the aptamer variant F30-2xdBroccoli (SEQ ID NO:1) was generated by PCR. By cutting the PCR01 amplicon with the restriction enzymes BamHI and EcoRI (Thermo Fisher Scientific), suitable overhangs were generated for the linearized plasmid pVAXl. Ligation integrated the PCR01 amplicon into the linearized plasmid backbone pVAXl.Plasmids that had successfully integrated the amplicon were identified by C012403-W001 / Gr.

[0217] A control restriction with avail was identified. The integrated sequence was verified by Sanger sequencing using the oligonucleotide T7-Prom (SEQ ID NO: 5). The resulting reporter plasmid was designated pVAXl F30-2xdBroccoli (SEQ ID NO: 6). This plasmid carries the F30-2xdBroccoli expression cassette with SEQ ID NO: 25. The expression cassette includes the promoter apFAB306 (SEQ ID NO: 26), the nucleic acid sequence (SEQ ID NO: 27) encoding the fluorogen-activating RNA aptamer F30-2xdBroccoli, and the terminator BBa-B1006 (SEQ ID NO: 28).

[0218] The strain E. coli WCM105 EfliC EyddS EendA was transformed using the RbCl method with the aptamer reporter plasmid pVAXl F30-2xdBroccoli, applied to an LB agar plate supplemented with 40 pM DFHBI-1T and 50 mg / L kanamycin sulfate, and then incubated at 37 °C for 16 hours. The following day, the culture plate was examined using the Alexa 488 application in a ChemiDoc MP Imaging System (Bio-Rad). Plasmid-bearing bacterial colonies could be distinguished from untransformed bacterial colonies or colonies transformed with the control plasmid pVAXl by the formation of a detectable fluorescence signal. Figure 2 shows the following bacterial cell populations: (A) E. coli WCM105 EfliC EyddS EendA after transformation with the reporter plasmid pVAXl F30-2xdBroccoli, cultured on LB agar with DFHBI-1T. Fluorescence emission is detectable in this bacterial cell colony. (B) untransformed E.E. coli WCM105 EfliC EyddS EendA bacterial colonies cultured on LB agar with DFHBI-1T. (C) E. coli WCM105 EfliC EyddS EendA bacterial colonies transformed with the control plasmid pVAXl and cultured on LB agar with DFHBI-1T. The bacterial cell colonies in both samples (B) and (C) show no detectable changes.

[0219] Fluorescence emission. C012403-W001 / Gr

[0220] 53

[0221] Example 2. Detection and quantification of plasmid loss rates

[0222] The strain E. coli WCM105 EfliC EyddS EendA was transformed on day 1 using the RbCl method with the aptamer reporter plasmid pVAXl F30-2xdBroccoli (SEQ ID NO: 6) (WCM105 EfliC EyddS EendA x pVAXl F30-2xdBroccoli), applied to an LB agar plate supplemented with 50 mg / L kanamycin sulfate, and then incubated at 37 °C for 16 hours. In parallel, the strain E. coli WCM105 EfliC EyddS EendA was applied to an unsupplemented LB agar plate without prior transformation and then incubated at 37 °C for 16 hours. On the following day (day 2), 2 mL of LB medium supplemented with 50 mg / L kanamycin sulfate was inoculated with WCM105 EfliC EyddS EendA x pVAXl F30-2xd broccoli and incubated for 12 hours at 37 °C. In parallel, 2 mL of unsupplemented LB medium was inoculated with WCM105 EfliC EyddS EendA and incubated for 12 hours at 37 °C.On the following day (day 3), WCM105 EfliC EyddS EendA x pVAXl F30-2xdBroccoli was inoculated in parallel into 2 mL of LB medium supplemented with 50 mg / L kanamycin sulfate and into 2 mL of unsupplemented LB medium. WCM105 EfliC EyddS EendA was also inoculated into 2 mL of unsupplemented LB medium. All cultures were incubated again for 12 hours at 37 °C. On the following day (day 4), the optical density at 600 nm (ODeoo) was determined for all cultures, and 1 mL of PBS and 1 mL of PBS supplemented with 40 pM DFHBI-1T were inoculated to an optical density of ODeoo = 0.02. Subsequently, all samples were measured using the Gell Counter Cytoflex SRT (Beckmann Coulter), and the fluorescence emission was determined at the single-cell level. At least 10,000 events were recorded for each sample. The culture of E. coli WCM105 EfliC EyddS EendA served as a control for non-plasmid-bearing bacterial cells.The fluorescence emission of the bacterial cells in this culture corresponds to the fluorescence emission of non-plasmid-bearing bacterial cells. All bacterial cells of the remaining C012403-W001 / Gr.

[0223] 54

[0224] Cultures exhibiting a fluorescence emission greater than the maximum measured for E. coli WCM105 Efli C EyddS EendA were identified as plasmid-carrying. All bacterial cells exhibiting a fluorescence emission less than or equal to the maximum measured for E. coli WCM105 Efli C EyddS EendA were identified as non-plasmid-carrying (Figure 3, left figure). The percentage plasmid loss rate was calculated by dividing the number of non-plasmid-carrying bacterial cells in the WCM105 Efli C EyddS EendA x pVAXl F30-2xdBroccoli cultures by the total number of events recorded for that sample (10,000).In parallel with the determination of fluorescence emission and the calculation of the plasmid loss rate, cultures of WCM105 Efli C EyddS EendA x pVAXl F30-2xd broccoli were inoculated into 2 mL of LB medium supplemented with 50 mg / L kanamycin sulfate and into 2 mL of unsupplemented LB medium and incubated for 12 hours at 37 °C. Additionally, the culture of WCM105 Efli C EyddS EendA was inoculated into 2 mL of unsupplemented LB medium and incubated for 12 hours at 37 °C. On the following day (day 5), the determination of fluorescence emission at the single-cell level was performed as described for day 4, the plasmid loss rate was determined (Figure 3, right-hand figure), and the bacterial cultures were inoculated as previously described.The steps performed on day 5 were repeated on all subsequent days until a plasmid loss rate of over 90% was observed in the culture of WCM105 Efli C EyddS EendA x pVAXl F30-2xd broccoli in unsupplemented LB medium. The plasmid loss rate was calculated by dividing the plasmid loss rate by the cultivation time. C012403-W001 / Gr.

[0225] 55

[0226] Example 3. Quantification of the specific plasmid titer

[0227] Production of the aptamer reporter plasmid pVAXl T112C-F30- 2xdBroccoli :

[0228] Using the aptamer reporter plasmid pVAXl F30-2xdBroccoli (SEQ ID NO: 6) as a template and the oligonucleotides Pp737 (SEQ ID NO: 7) and Pp732 (SEQ ID NO: 8), the 472 base pair amplicon PCR02 (SEQ ID NO: 9) was generated by PCR. Using the plasmid pBR322 as a template and the oligonucleotides Pp729 (SEQ ID NO: 10) and Ppl30 (SEQ ID NO: 11), the 500 base pair amplicon PCR03 (SEQ ID NO: 12), containing the RNAI / RNAII overlap region of the pMBl origin of replication, was generated by PCR. By Overlap-extension-PCR (Horton et al. 2013, Gene Splicing by Overlap Extension: Tailor- Made Genes Using the Polymerase Chain Reaction, BioTechniques 54, 129-33) using the oligonucleotides Pp737 and Ppl30, the amplicons PCR02 and PCR03 were combined to form a combined amplicon PCR04 (SEQ ID NO: 13).The plasmid pVAXl F30-2xdBroccoli was linearized by cutting with the restriction enzymes Nael and ApaLI (Thermo Fisher Scientific), and a 2470 base pair fragment was isolated by excision from an agarose gel. The amplicon PCR04 was integrated into the linearized plasmid pVAXl F30-2xdBroccoli by infusion cloning (Takara Bio). The integrated sequence was verified by Sanger sequencing using the oligonucleotide pVAX-seq23-fw (SEQ ID NO: 14). The resulting reporter plasmid was designated pVAXl T112C-F30-2xdBroccoli (SEQ ID NO: 15).

[0229] The strain E. coli WCM105 EfliC HyddS HendA was transformed individually with the control plasmid pVAXl and the aptamer reporter plasmids pVAXl F30-2xdBroccoli and pVAXl T112C- F30-2xdBroccoli using the RbCl method; the resulting cultures were separately transferred to LB-C012403-W001 / Gr supplemented with 50 mg / L kanamycin sulfate.

[0230] 56

[0231] Agar plates were applied and then incubated at 37 °C for 16 hours. On the following day (day 1), 2 mL of LB medium supplemented with 50 mg / L kanamycin sulfate were individually inoculated with WCM105 EfliC EyddS EendA x pVAXl, WCM105 EfliC EyddS EendA x pVAXl F30-2xdBroccoli, and WCM105 EfliC EyddS EendA x pVAXl T112C- F30-2xdBroccoli and incubated for 12 hours at 37 °C. On the following day (day 2), the optical density at 600 nm (ODeoo) was determined for all cultures, and 1 mL of BBS and 1 mL of PBS supplemented with 40 pM DFHBI-1T were inoculated to an optical density of ODeoo = 0.02. Subsequently, all samples were measured on the Gell Counter Cytoflex SRT (Beckmann Coulter), and 10,000 events were recorded for each sample. Figure 4 shows the determination of the fluorescence emission of E. coli WCM105 EfliC EyddS EendA, transformed with pVAXl, and of E. coli WCM105 EfliC EyddS EendA, transformed with pVAXl, and of E.E. coli WCM105 EfliC EyddS EendA, transformed with pVAXl T112C F30-2xdBroccoli, is shown. The culture of E. coli WCM105 EfliC EyddS EendA x pVAXl served as a control for bacterial cells without aptamer reporters. The culture of E. coli WCM105 EfliC EyddS EendA x pVAXl F30-2xdBroccoli served as a control for bacterial cells transformed with a plasmid with an unchanged plasmid copy number. The culture of E. coli WCM105 EfliC EyddS EendA x pVAXl F30-2xdBroccoli served as a sample for bacterial cells transformed with a plasmid with an altered and unknown plasmid copy number. The mean fluorescence emission was determined for all cultures using the statistics function of the CytoFlex software. Figure 5 shows the mean fluorescence emission of E. coli WCM105 EfliC EyddS EendA, transformed with pVAXl, pVAXl F30-2xdBroccoli or pVAXl T112C F30-2xdBroccoli, as determined using the statistics function of the CytExpert SRT software. C012403-W001 / Gr.

[0232] 57

[0233] By comparing the fluorescence emission measured in DBS supplemented with 40 pM DFHBI-1T, the relative plasmid copy number of the plasmids pVAXl F30-2xdBroccoli and pVAXl T112C-F30-2xdBroccoli in bacterial cells of WCM105 fli C l yddS t endA was determined. This revealed that pVAXl T112C-F30-2xdBroccoli has a lower plasmid copy number than pVAXl F30-2xdBroccoli. Using the Gene JET Plasmid Miniprep Kit (Thermo Fisher Scientific), plasmid DNA was isolated from all cultures, and the concentration in the elution fraction was subsequently determined by UV / VIS spectrometry. To calculate the plasmid titer in the respective cultures, the measured plasmid concentration was first multiplied by the elution volume and then divided by the volume of the culture volume used for sampling.To calculate the specific titer, the plasmid titer was divided by the optical density of the cultures used for plasmid isolation at 600 nm. Figure 6 shows the specific plasmid titer [mg / L / ODeoo] of the plasmids pVAXl T112C F30-2xdBroccoli and pVAXl F30-2xdBroccoli in shake flask culture.

[0234] The increased fluorescence emission in bacterial cells transformed with pVAXl F30-2xdBroccoli compared to those transformed with pVAXl T112C-F30-2xdBroccoli indicates the higher average plasmid copy number of pVAXl F30-2xdBroccoli compared to pVAXl T112C-F30-2xdBroccoli (see Figure 5), and thus allows the prediction of an increased specific plasmid titer in cultures of cells transformed with pVAXl F30-2xdBroccoli compared to those transformed with pVAXl T112C-F30-2xdBroccoli. This prediction was confirmed by determining the plasmid titer in the samples used for investigation by plasmid isolation (see Figure 6). The method shown is therefore suitable for C012403-W001 / Gr

[0235] 58 reliable quantification of the plasmid copy number and prediction of the expected specific plasmid titer.

[0236] Example 4: Quantification of the plasmid copy number

[0237] Population level

[0238] The strain E. coli WCM105 EfliC EyddS EendA was transformed individually with the control plasmid pVAXl and the aptamer reporter plasmids pVAXl F30-2xdBroccoli and pVAXl T112C- F30-2xdBroccoli using the RbCl method, the resulting cultures were applied separately to LB agar plates supplemented with 50 mg / L kanamycin sulfate and then incubated at 37 °C for 16 hours. On the following day (day 1), 2 mL of LB medium supplemented with 50 mg / L kanamycin sulfate at were individually inoculated with WCMIO 5 EfliC EyddS EendA x pVAXl, WCMIO 5 EfliC EyddS EendA x pVAXl F30-2xdBroccoli, and WCM105 EfliC EyddS EendA x pVAXl T112C- F30-2xdBroccoli and incubated for 12 hours at 37 °C. On the following day (day 2), all cultures were transferred at a ratio of 1:100 into 2 mL of fresh LB medium supplemented with 50 mg / L kanamycin sulfate at and cultured for six hours at 37 °C.Subsequently, the optical density at 600 nm (ODeoo) was determined for all cultures. Bacterial cells were sedimented from 1 mL of each culture by centrifugation at 5000 x g for 4 min, and the liquid supernatant was discarded. The sedimented bacterial cells were then suspended in 1 mL of PBS. The resulting suspensions were each divided into two 500 pL aliquots. The suspended bacterial cells were sedimented by centrifugation as described above, and the liquid supernatant was discarded. Bacterial cells from the first of the previously prepared aliquots were then suspended in 500 pL of PBS, and bacterial cells from the second of the previously prepared aliquots were suspended in 500 pL of C012403-W001 / Gr. supplemented with 40 pM DFHBI-1T.

[0239] 59

[0240] PBS was suspended. 100 pl of the resulting suspensions were transferred to 96-well plates (Greiner Sensoplate Microplate, 96 Well, PS, F-Bottom, Glass Bottom, Black, Lid, Sterile) for fluorescence emission determination and measured on a Biotek Synergy HL (Agilent) (X ex = 472 nm, X em= 507 nm). PBS supplemented with 40 pM DFHBI-1T served as a control for determining the background fluorescence. For normalization, the determined fluorescence emission was subsequently divided by the previously determined optical density at 600 nm to determine the mean fluorescence emission. Figure 7 shows the mean fluorescence emission of E. coli WCM105 EfliC EyddS AendA_bacterial cell populations transformed with pVAXl, pVAXl F30-2xdBroccoli, or pVAXl T112C F30-2xdBroccoli, as determined using a Biotek Synergy Hl plate reader (Agilent). The increased fluorescence emission of bacterial cell populations transformed with pVAXl F30-2xdBroccoli compared to bacterial cell populations transformed with pVAXl T112C-F30-2xdBroccoli shows the higher average plasmid copy number of pVAXl F30-2xdBroccoli compared to pVAXl T112C-F30-2xdBroccoli.The method shown is therefore suitable for reliably determining the plasmid copy number and predicting the expected specific plasmid titer.

[0241] Example 5. Screening for bacterial cell populations with higher specific plasmid titer

[0242] Using the aptamer reporter plasmid pVAXl T112C F30-2xdBroccoli as a template (SEQ ID NO: 15) and the oligonucleotides Pp737 (SEQ ID NO: 07) and Pp900 (SEQ ID NO: 16), the 711 base pair amplicon PCR05 (SEQ ID NO: 17) was generated by PCR. This amplicon contains the RNAI / RNAII overlap region of the pMBl origin of replication, as well as three degenerate positions immediately 3' of the RNAI / RNAII overlap region. (C012403-W001 / Gr)

[0243] Using the aptamer reporter plasmid pVAXl T112C F30- 2xdBroccoli as a template and the oligonucleotides Pp901 (SEQ ID NO: 18) and Pp740 (SEQ ID NO: 19), the 575 base pair long amplicon PCR06 (SEQ ID NO: 20) containing the RNAII region of the pMBl origin of replication was generated by PCR. The resulting amplicons were subsequently combined by overlap extension PCR (Horton et al. 2013, Gene Splicing by Overlap Extension: Tailor-Made Genes Using the Polymerase Chain Reaction, BioTechniques 54, 129-33) using the oligonucleotides Pp737 and Pp740 to form a combined amplicon PCR07 (SEQ ID NO: 21), and this was inserted into the plasmid pVAXl T112C F30-2xdBroccoli, which had been cut with the restriction enzymes Nael and NruI (Thermo Fisher Scientific), by ligation with T4 ligase (Thermo Fisher Scientific). The resulting plasmid library was designated pVAXl T112C-F30-2xdBroccoli lib (SEQ ID NO: 22).

[0244] The strain E. coli WCM105 EfliC EyddS EendA was transformed individually with the aptamer reporter plasmid pVAXl T112C F30-2xdBroccoli and the plasmid library pVAXl T112C F30-2xdBroccoli lib using the RbCl method. The resulting transformants E. coli WCM105 EfliC EyddS EendA pVAXl T112C F30-2xdBroccoli and E. coli WCM105 EfliC EyddS EendA pVAXl T112C F30-2xdBroccoli lib were initially incubated for one hour at 37 °C in unsupplemented LB medium. Subsequently, cultures were prepared by over-inoculation in 2 mL of LB medium supplemented with 50 mg / L kanamycin sulfate and incubated for 12 hours at 37 °C. On the following day (day 2), the cultures were measured as previously described using the Cell Counter Cytoflex SRT (Beckmann Coulter). The culture of E. coli WCM105 EfliC EyddS EendA pVAXl T112C F30-2xdBroccoli served as a control for bacterial cells with an unaltered plasmid copy number. Bacterial cells from the culture of E.coli WCM105 EfliC EyddS EendA C012403-W001 / Gr.

[0245] 61 pVAXl T112C F30-2xdBroccoli lib cells, which exhibited a fluorescence emission higher than the maximum fluorescence emission determined for cells of the culture of E. coli WCM105 EfliC EyddS EendA pVAXl T112C F30-2xdBroccoli (Figure 8; PI), were interpreted as bacterial cells of a bacterial cell population with an elevated specific titer, transferred by tube sorting into 500 pL of LB medium supplemented with 50 mg / L kanamycin sulfate, incubated at 37 °C for five hours, and subsequently plated onto LB agar plates supplemented with 50 mg / L kanamycin sulfate. On the following day (day 3), individual colonies were used for inoculation with 2 mL each of LB medium supplemented with 50 mg / L kanamycin sulfate, and the resulting cultures were incubated for 16 hours at 37 °C. In parallel, E.coli WCM105 EfliC EyddS EendA pVAXl T112C F30-2xdBroccoli was inoculated in 2 mL of LB medium supplemented with 50 mg / L kanamycin sulfate, and the resulting culture was incubated for 16 hours at 37 °C. On the following day (day 4), plasmid DNA was isolated from all previously prepared cultures using the Gene JET Plasmid Miniprep Kit (Thermo Fisher Scientific), and the plasmid concentration in the elution fraction was determined by UV / VIS spectrometry. The plasmid titer was calculated by multiplying the measured plasmid concentration by the elution volume and then dividing by the culture volume used. The culture of E.coli WCM105 EfliC EyddS EendA pVAXl T112C F30-2xdBroccoli was used as a control for the isolation of plasmids with an unaltered plasmid copy number and an unaltered specific plasmid titer.The sequences of the replicons of plasmids exhibiting an elevated specific plasmid titer compared to pVAXl T112C F30-2xdBroccoli were determined by sequencing with the oligonucleotide pVAX-seq23-fw (SEQ ID NO: 14). All plasmids exhibiting an elevated specific plasmid titer compared to pVAXl T112C F30-2xdBroccoli showed C012403-W001 / Gr base exchanges within the degenerate region, but not at other positions of replication origin. Two isolates, 5bLl (SEQ ID NO:23) and 6bRl (SEQ ID NO:24), which exhibited an elevated specific plasmid titer, as well as the control plasmid pVAXl T112C F30-2xdBroccoli, were used to transform E. coli WCM105 EfliC EyddS EendA. The resulting transformants were incubated in LB medium supplemented with 50 mg / L kanamycin sulfate for 16 hours at 37 °C.Plasmid DNA was then isolated from the cultures using the Gene JET Plasmid Miniprep Kit (Thermo Fisher Scientific), and the plasmid concentration in the elution fraction was determined by UV / VIS spectrometry. To calculate the plasmid titer in the respective cultures, the measured plasmid concentration was first multiplied by the elution volume and then divided by the volume of the culture used for sampling. To calculate the specific titer, the plasmid titer was divided by the optical density of the cultures used for plasmid isolation at 600 nm. In cultures transformed with the plasmid variants isolated by screening, an increased specific plasmid titer was again found compared to pVAXl T112C F30-2xdBroccoli (see Figure 9), thus confirming the successful isolation of replicon variants with a stably increased plasmid copy number compared to the original variant.The method shown is therefore suitable for the reliable isolation of plasmids that produce a higher specific plasmid titer in bacterial cell culture compared to the original variant. C012403-W001 / Gr.

[0246] The present invention is further described by the following points.

[0247] 1. In vivo method for comparing the specific plasmid titers in two or more bacterial cell populations using RNA aptamers, wherein the method comprises: a) providing one or more different plasmids, wherein the plasmid or the several different plasmids each comprise an identical expression cassette comprising a promoter functionally linked to a nucleic acid sequence and a terminator linked to the 3' end of the nucleic acid sequence, wherein the nucleic acid sequence exclusively codes for a molecule selected from the group of fluorogen-activating RNA aptamers; b) transforming each bacterial cell population with one plasmid from a); c) cultivating each bacterial cell population under defined cultivation conditions;d) Contacting each bacterial cell population with a binding partner for the fluorogen-activating RNA aptamer, whereby binding of the fluorogen-activating RNA aptamer expressed by the bacterial cells with the binding partner generates a fluorescence signal; and e) Detecting the fluorescence signal of the bacterial cells of the individual bacterial cell populations; f) Comparing the fluorescence signals of the bacterial cells of the individual bacterial cell populations, whereby a stronger fluorescence signal indicates a higher specific plasmid titer in the respective bacterial cell population.

[0248] 2. In vivo procedure: Procedure according to point 1, further comprising step el) normalizing the fluorescence signal, wherein in step f) comparing the normalized fluorescence signals of C012403-W001 / Gr

[0249] 64

[0250] Bacterial cells of the individual bacterial cell populations are analyzed, with a stronger normalized fluorescence signal indicating a higher specific plasmid titer in the respective bacterial cell population.

[0251] 3. In vivo procedure according to point 2, wherein the normalization of the fluorescence signal is carried out with regard to the number of bacterial cells in the bacterial cell population.

[0252] 4. In vivo procedure according to one of points 1-3, in which the individual bacterial cell populations are each transformed with a different plasmid.

[0253] 5. In vitro procedure according to one of points 1-4, wherein the two or more different bacterial cell populations are genotypically different.

[0254] 6. In vivo procedure according to one of points 1-5, wherein the cultivation of the individual bacterial cell populations takes place under different cultivation conditions.

[0255] 7. A method according to one of points 1 to 5, wherein the cultivation of the individual bacterial cell populations takes place under substantially the same cultivation conditions.

[0256] 8. Method according to one of points 1 to 7, wherein the cultivation of the individual bacterial cell populations is carried out in a selective medium.

[0257] 9. Method according to any one of points 1 to 7, wherein the cultivation of the individual bacterial cell populations is carried out under non-selective conditions. C012403-W001 / Gr

[0258] 65

[0259] 10. A method according to any of the preceding points, wherein the fluorogen-activating RNA aptamer is selected from the group consisting of Spinach aptamer, Spinach2 aptamer, F30-2xdBroccoli aptamer, Broccoli aptamer, Red Broccoli aptamer, Orange Broccoli aptamer, Corn aptamer, Beetroot aptamer, Mango, Mango II, Mango III, Mango IV, Malachite Green aptamer and derivatives thereof, preferably from the group consisting of Spinach2 aptamer, F30-2xdBroccoli aptamer and Broccoli aptamer.

[0260] 11. Method according to any of the preceding points, wherein the bacterial cell is permeable to the binding partner.

[0261] 12. Method according to any of the preceding points, wherein the binding partner is selected from the group consisting of DFHBI, DFHBI-1T, DFHO, DMHBI, DFAME and TOl-Biotin, and preferably is DFHBI-1T.

[0262] 13. Method according to any of the preceding points, wherein the fluorescence-inducing RNA aptamer is selected from F30-2xdBroccoli aptamer and Broccoli aptamer, and wherein the binding partner is DFHBI-1T.

[0263] 14. In vivo procedure to compare the specific plasmid titers according to one of the preceding steps, further comprising step g) selecting the bacterial cell populations which exhibit the strongest fluorescence signal.

[0264] 15. In vivo method according to point 1 for comparing the specific plasmid titers in two or more genotypically identical bacterial cell populations, using RNA aptamers, wherein the method comprises: C012403-W001 / Gr

[0265] 66 a) Providing two or more different plasmids, wherein the different plasmids each comprise the same expression cassette, comprising a promoter functionally linked to a nucleic acid sequence and a terminator linked to the 3' end of the nucleic acid sequence, wherein the nucleic acid sequence exclusively codes for a molecule selected from the group of fluorogen-activating RNA amino acids; b) Transforming each bacterial cell population with each of the different plasmids from a); c) Cultivating each bacterial cell population under substantially the same cultivation conditions;d) Contacting each bacterial cell population with the same binding partner for the fluorogen-activating RNA aptamer, whereby binding of the fluorogen-activating RNA aptamer expressed by the bacterial cells to the binding partner generates a fluorescence signal; and e) Detecting the fluorescence signal of the bacterial cells of the individual bacterial cell populations; f) Comparing the fluorescence signals of the bacterial cells of the individual bacterial cell populations, whereby a stronger fluorescence signal indicates a higher specific plasmid titer in the respective bacterial cell population.

[0266] 16. In vivo method according to point 1 for comparing the specific plasmid titers in two or more genotypically distinct bacterial cell populations, using RNA aptamers, wherein the method comprises: a) providing a plasmid, wherein the plasmid comprises an expression cassette comprising a promoter functionally associated with a nucleic acid sequence and a terminator associated with the 3' end of the nucleic acid sequence C012403-W001 / Gr

[0267] 67 is connected, wherein the nucleic acid sequence exclusively codes for a molecule selected from the group of fluorogen-activating RNA aptamers; b) Transforming the individual bacterial cell populations with the plasmid from a); c) Cultivating the individual bacterial cell populations each under substantially the same cultivation conditions; d) Contacting the individual bacterial cell populations each with the same binding partner for the fluorogen-activating RNA aptamer, whereby a fluorescence signal is generated by binding of the fluorogen-activating RNA aptamer expressed by the bacterial cells with the binding partner;and e) detecting the fluorescence signal of the bacterial cells of the individual bacterial cell populations, f) comparing the fluorescence signals of the bacterial cells of the individual bacterial cell populations, where a stronger fluorescence signal indicates a higher specific plasmid titer in the bacterial cell population in question.

[0268] 17. In vivo method according to point 1 for comparing the specific plasmid titers in two or more identical bacterial cell populations, using RNA aptamers, wherein the method comprises: a) providing a plasmid, wherein the plasmid comprises an expression cassette comprising a promoter functionally linked to a nucleic acid sequence and a terminator linked to the 3' end of the nucleic acid sequence, wherein the nucleic acid sequence exclusively codes for a molecule selected from the group of fluorogen-activating RNA aptamers; b) transforming the individual bacterial cell populations with the plasmid from a); C012403-W001 / Gr

[0269] 68 c) Cultivating the individual bacterial cell populations each under different cultivation conditions; d) Contacting the individual bacterial cell populations each with the same binding partner for the fluorogen-activating RNA aptamer, whereby binding of the

[0270] Bacterial cells expressing fluorogen-activating RNA aptamer with the binding partner generate a fluorescence signal; and e) detecting the fluorescence signal of the bacterial cells of the individual bacterial cell populations, f) comparing the fluorescence signals of the bacterial cells of the individual bacterial cell populations, where a stronger fluorescence signal indicates a higher specific plasmid titer in the bacterial cell population in question.

Claims

C012403-W001 / Gr 1 Patentansprüche 1. An in vivo method for comparing the specific plasmid titers in two or more genotypically identical bacterial cell populations using RNA aptamers, wherein the method comprises: a) providing two or more different plasmids, wherein the different plasmids each comprise an identical expression cassette comprising a promoter functionally linked to a nucleic acid sequence and a terminator linked to the 3' end of the nucleic acid sequence, wherein the nucleic acid sequence exclusively encodes a molecule selected from the group of fluorogen-activating RNA aptamers; b) transforming each bacterial cell population with each of the different plasmids from a); c) cultivating each bacterial cell population under substantially identical cultivation conditions;d) Contacting each bacterial cell population with the same binding partner for the fluorogen-activating RNA aptamer, whereby binding of the fluorogen-activating RNA aptamer expressed by the bacterial cells to the binding partner generates a fluorescence signal; and e) Detecting the fluorescence signal of the bacterial cells of the individual bacterial cell populations, wherein the detection of the fluorescence signal is possible in vivo without requiring lysis of the bacterial cells; f) Comparing the fluorescence signals of the bacterial cells of the individual bacterial cell populations, wherein a stronger fluorescence signal indicates a higher specific plasmid titer in the respective bacterial cell population. C012403-W001 / Gr 2. The method of claim 1, wherein the detection of the fluorescence signal in step e) is carried out by means of flow cytometric analysis (FACS) or by means of analysis in plate readers.

3. In vitro method according to claim 1 or 2, further comprising step el) normalizing the fluorescence signal, wherein in step f) the normalized fluorescence signals of the bacterial cells of the individual bacterial cell populations are compared, wherein a stronger normalized fluorescence signal indicates a higher specific plasmid titer in the bacterial cell population in question.

4. In vitro method according to claim 3, wherein the normalization of the fluorescence signal is performed with respect to the number of bacterial cells in the bacterial cell population.

5. Method according to one of the preceding claims, wherein the cultivation of the individual bacterial cell populations is carried out in a selective medium.

6. Method according to one of the preceding claims, wherein the cultivation of the individual bacterial cell populations is carried out under non-selective conditions.

7. A method according to any one of the preceding claims, wherein the fluorogen-activating RNA aptamer is selected from the group consisting of Spinach aptamer, Spinach2 aptamer, F30-2xdBroccoli aptamer, Broccoli aptamer, Red Broccoli aptamer, Orange Broccoli aptamer, Corn aptamer, Beetroot aptamer, Mango, Mango II, Mango III, Mango IV; Malachite Green aptamer, Cobalamin-binding aptamer and derivatives thereof, preferably from the group C012403-W001 / Gr 3 consisting of Spinach2-aptamer, F30-2xdBroccoli-aptamer and Broccoli-aptamer.

8. Method according to any of the preceding claims, wherein the bacterial cell is permeable to the binding partner.

9. Method according to any of the preceding claims, wherein the binding partner is selected from the group consisting of DFHBI, DFHBI-1T, DFHO, DMHBI, DFAME and TOl-Biotin, and preferably is DFHBI-1T.

10. Method according to any of the preceding claims, wherein the fluorescence-inducing RNA aptamer is selected from F30-2xdBroccoli aptamer and Broccoli aptamer, and wherein the binding partner is DFHBI-1T.

11. In vivo method for comparing the specific plasmid titers according to any of the preceding claims, further comprising step g) selecting the bacterial cell populations which exhibit the strongest fluorescence signal.

Images