Escherichia coli promoter screening system and screening method based on separated green fluorescent protein

Abstract

The invention relates to the field of molecular biology and protein engineering, in particular to an escherichia coli promoter screening system and screening method based on separated green fluorescent protein. According to the system, a single-plasmid high-throughput screening platform is constructed by utilizing a fluorescent recombination principle of separated GFP (Green Fluorescent Protein). The platform comprises a promoter library which can simultaneously express a target protein-GFP11 fragment and a GFP1-10 fragment in a single plasmid. When the expression level of the target protein is high, the GFP11 and the GFP1-10 can be recombined to form a complete GFP, and a fluorescence signal is generated. By measuring the ratio of the fluorescence intensity to the thallus concentration, the optimized promoter suitable for efficient soluble expression of the target protein can be efficiently screened out. The method is simple to operate, low in cost and high in throughput screening, and can be widely applied to the fields of protein expression condition optimization and gene engineering.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular biology and protein engineering, in particular to an Escherichia coli promoter screening system based on split green fluorescent protein and a screening method. BACKGROUND

[0002] Escherichia coli is one of the most commonly used prokaryotic protein expression systems. Among them, BL21(DE3) is a widely used expression host strain, and pET series plasmids are the most commonly used expression vectors. These plasmids usually carry a strong promoter of T7 RNA polymerase, which can be induced by IPTG to express the target gene. Due to the high expression strength of the T7 promoter, in many cases it will lead to the accumulation of a large amount of target protein in the host cell, forming insoluble inclusion bodies, which cannot be correctly folded and obtain biological activity.

[0003] Overexpression is often a major reason why proteins cannot be expressed at a high level of solubility in Escherichia coli. Even if optimization strategies such as co-expression of molecular chaperones, adjustment of induction conditions, etc. are taken, it is difficult to completely solve this problem. On the contrary, moderate regulation of the expression strength of the target gene, keeping it within a reasonable "soluble expression window" range, is conducive to the correct folding and soluble expression of the protein. Therefore, finding a suitable weak / medium strength promoter to replace the overly strong T7 promoter is crucial for improving the soluble expression level of the protein.

[0004] In response to the technical demand of improving the soluble expression level of the target protein by regulating the expression strength, there have been some related research progress and attempts. The prior art discloses an Escherichia coli promoter library and its application. The patent constructs a plasmid library containing more than 1800 synthetic promoters of different strengths, and predicts the expression strength of each promoter through deep sequencing and computer algorithm. Alper et al. published a study in 2005 Nat Methods, which used whole genome resequencing technology to identify hundreds of new transcription start sites on the Escherichia coli genome, providing a new resource library for promoter screening. Mutalik et al. published a study in 2019 Nat Methods, which developed an automated platform to intelligently design and optimize synthetic promoters and RBS sequences through machine learning algorithms to achieve precise regulation of target gene expression in Escherichia coli. However, the above-mentioned promoter libraries do not have a matching high-throughput method for screening, especially for soluble expression level screening.

[0005] Green fluorescence protein (GFP) is the most widely used fluorescent protein. GFP is formed by 11 beta-sheet cycles arranged in a barrel structure, and the alpha-helix containing the chromophore is contained inside the barrel. US7198924B2 discloses a method for large-scale protein expression screening using green fluorescent protein (GFP) as a reporter molecule. The target gene is expressed in fusion with GFP, and the GFP fluorescence intensity can reflect the protein expression level. Makoff et al. first used GFP as an active reporter to identify promoter strength in E. coli in their research published in J Gen Microbiol in 1989. Cheah et al. used fluorescent protein as a reporter gene to identify gene sequences suitable for high expression in E. coli by high-throughput gene synthesis and expression screening in their research published in Mol Biosyst in 2013. However, there are problems in using GFP alone for screening, such as inconsistent expression intensity of different target genes with GFP.

[0006] In general, there is a certain research foundation and patent reserve for E. coli promoter screening, GFP and its derivatives in protein expression analysis, but the related research on the organic combination of these technologies and the development of an efficient expression condition screening platform is still relatively weak. The present application provides an innovative solution to meet this demand. SUMMARY

[0007] Therefore, the present application provides an E. coli promoter screening system and method based on isolated green fluorescent protein. The system and method can efficiently screen an optimized promoter suitable for high-efficiency and soluble expression of a target protein.

[0008] To achieve the above-mentioned purposes, the present application provides the following technical solutions:

[0009] In a first aspect, the present application provides an E. coli promoter screening system based on isolated green fluorescent protein, comprising:

[0010] (I) a promoter library, wherein the promoter library comprises a plurality of promoters with different strengths;

[0011] (II) a target gene-GFP11 fragment, wherein the target gene and the GFP11 fragment are connected by a peptide group;

[0012] (III) a tac promoter-driven GFP1-10 fragment;

[0013] (IV) a plasmid vector, wherein the plasmid vector comprises a plurality of enzyme digestion sites for cloning the promoter library, the target gene-GFP11 fragment and the GFP1-10 fragment.

[0014] In some embodiments of the present application, the promoter library is from 20 different strength E. coli promoters of iGEM, each of which is connected with EcoRI and HindIII enzyme cutting sites upstream and downstream, respectively.

[0015] In some embodiments of the present application, the peptide group has:

[0016] (I) an amino acid sequence as shown in SEQ ID No. 22;

[0017] (II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence as described in (I), and an amino acid sequence functionally identical to the amino acid sequence as described in (I); or

[0018] (III) an amino acid sequence having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% or more identity to the amino acid sequence as described in (I) or (II).

[0019] In some embodiments of the present application, the upstream of the target gene-GFP11 fragment is a HindIII enzyme cutting site, and the downstream is a BamHI enzyme cutting site; and / or

[0020] The upstream of the GFP1-10 fragment is an EcoRI enzyme cutting site, and the downstream is a BamHI enzyme cutting site;

[0021] The single-plasmid vector comprises pET-28a(+).

[0022] In a second aspect, the present application further provides a host transformed with the E. coli promoter screening system.

[0023] 6. The host according to claim 5, comprising E. coli.

[0024] Preferably, the strain of E. coli comprises BL21(DE3).

[0025] In a third aspect, the present application further provides an application of any of the following in screening promoters:

[0026] (I) the E. coli promoter screening system; or

[0027] (II) the host.

[0028] In a fourth aspect, the present application further provides a method for screening E. coli promoters using the E. coli promoter screening system, comprising the following steps:

[0029] a) constructing the promoter library, the target gene-GFP11 fragment and the GFP1-10 fragment; cloning into the plasmid vector;

[0030] b) transforming the constructed plasmid vector into an E. coli expression host strain;

[0031] c) inducing expression of the target gene-GFP11 and GFP1-10 fragments;

[0032] d) measuring the fluorescence intensity and the bacterial concentration of each transformant;

[0033] e) calculating the ratio of the fluorescence intensity to the bacterial concentration, and screening the transformant with the highest ratio;

[0034] f) sequencing the transformant with the highest ratio to identify the promoter sequence carried by the transformant.

[0035] In some specific embodiments of the present application, the induction in step c) employs IPTG induction.

[0036] In a fifth aspect, the present application also provides a computer device, a computer readable storage medium or a computer program product,

[0037] The computer device comprises a memory and a processor, the memory stores a computer program, and the processor implements the steps of the method when executing the computer program; or

[0038] The computer readable storage medium stores a computer program, and the computer program is executed by a processor to implement the steps of the method; or the computer program product comprises a computer program, and the computer program is executed by a processor to implement the steps of the method.

[0039] The present application discloses an E. coli promoter high-throughput screening system based on split green fluorescent protein (split GFP) and an application method thereof. The system integrates a widely used promoter library with different expression intensities, a target gene fusion GFP11 reporter fragment and an endogenous GFP1-10 complementation fragment in a single plasmid vector, and can efficiently and economically screen an optimized promoter suitable for high-level soluble expression of a target protein.

[0040] The innovation of the present application mainly lies in:

[0041] 1. More than 20 classic E. coli synthetic promoters from public resources such as iGEM are collected, and the expression intensity thereof covers a wide range, providing rich candidate resources for screening.

[0042] 2. The split GFP technology is applied to the expression condition optimization field for the first time, and the soluble expression level of the target protein is reflected by the recombinant GFP as a reporter signal, avoiding the cumbersome process of traditional culture-lysis-detection.

[0043] 3. All necessary expression elements are integrated into a single plasmid system, without complex gene operation, facilitating construction and high-throughput screening, and being simple and short in operation.

[0044] 4. The actual application results show that the soluble expression level of the target protein of the recombinant strain optimized by the system is increased by more than 2.5 times compared with the original strain, the optimization effect is remarkable, and the system has a wide application prospect.

[0045] In summary, the present application provides an innovative solution for efficient optimization of recombinant protein expression conditions, and has universality, practicality and economy, and has important theoretical significance and application value in the fields of basic research and biopharmaceuticals. BRIEF DESCRIPTION OF DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows.

[0047] Figure 1 Figure 1 shows the expression level of gmd and SOD and the comparison of fluorescence / OD600 ratio; wherein a) the expression of gmd protein in E. coli, the left side is the culture condition at 25℃, and the right side is the culture condition at 37℃; M: protein molecular weight marker; W: whole cell lysate; "S" and "P" respectively represent the supernatant soluble protein and insoluble precipitate; the results show that gmd is expressed in soluble form at high efficiency under two temperatures; b) the expression of SOD protein in E. coli, the left side is the culture at 25℃, and the right side is the culture at 37℃, the labels are the same as a); the results show that SOD mainly accumulates in the form of insoluble inclusion body, and the soluble expression level is low; c) the soluble expression level of gmd and SOD is detected by using the split GFP screening system of the present application, the vertical axis is the fluorescence intensity / OD600 ratio of the strain, reflecting the soluble expression level of the target protein, the blue column represents the gmd expression strain, and the ratio is 185781; the green column represents the SOD expression strain, and the ratio is only 37565, which is about 5 times different from each other, which is consistent with the above gel electrophoresis results, verifying that the screening system can effectively distinguish different soluble target proteins;

[0048] Figure 2 Figure 2 shows a construction example of the screening plasmid; the promoter library, the target gene and the backbone are connected by EcoRI, HindIII and BamHI at one time to obtain an expression library containing different promoters;

[0049] Figure 3 Fluorescence intensity / OD of T7-P01 expression strain and optimal strain after screening 600 Ratio; the fluorescence / OD600 ratio of the strain optimized by the screening system of the application is about 2.57 times higher than that of the original T7-P01 strain;

[0050] Figure 4 SDS-PAGE of T7-P01 and J23118-P01 expression strains; wherein, the left side is the T7-P01 strain driven by the T7 promoter, and the right side is the J23118-P01 strain driven by the J23118 promoter obtained by screening, and each group has three repeats (1-3); each sample is divided into whole cell lysate ("whole"), supernatant soluble protein ("upper layer") and insoluble precipitate ("precipitate") for SDS-PAGE detection. DETAILED DESCRIPTION

[0051] The application discloses an Escherichia coli promoter screening system based on split green fluorescent protein and a screening method, and those skilled in the art can refer to the content herein and appropriately improve process parameters for implementation. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art, and they are all regarded as included in the application. The method and application of the application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of the application, to realize and apply the technical solution of the application.

[0052] The application discloses an Escherichia coli promoter screening system based on split green fluorescent protein (split GFP) and an application method thereof. The system integrates a promoter library, a target gene fusion, a GFP complementation and other modules, and can realize high-throughput screening of high-efficiency soluble expression conditions of the target protein.

[0053] 1. Composition structure of the system

[0054] a) Promoter library

[0055] The promoter library comprises a plurality of Escherichia coli promoter sequences with different intensities. The application preferably adopts 20 synthetic promoters with different intensities of iGEM, and each promoter is provided with EcoRI and HindIII enzyme cutting sites at two ends, so as to facilitate cloning operation. The promoter library can be expanded and optimized according to actual needs.

[0056] b) Target gene-GFP11 fusion expression element

[0057] The target gene is linked to the coding sequence of GFP11 by a GGGGSGGGGS short peptide sequence to form a fusion. The upstream is a HindIII restriction site and the downstream is a BamHI restriction site for cloning operation. The GFP11 fragment is about 16 amino acids long and can recombine with the GFP1-10 fragment to form a fluorescently active GFP.

[0058] c) Coding sequence of GFP1-10

[0059] The GFP1-10 fragment coding about 230 amino acids is driven by the E. coli tac promoter for expression. The upstream is an EcoRI restriction site and the downstream is a BamHI restriction site.

[0060] d) Single-plasmid co-expression vector

[0061] The present application selects pET-28a(+) as the backbone, which contains the corresponding restriction sites at the multiple cloning sites, and the three elements are integrated into a single plasmid by restriction and ligation, and the target gene-GFP11 and GFP1-10 are co-expressed in the same host strain.

[0062] 2. Working principle

[0063] When the above elements are co-expressed in the E. coli expression host, if the soluble expression level of the target protein is high, the GFP11 fragment will recombine with the GFP1-10 fragment to form a complete GFP, resulting in a detectable green fluorescence from the bacterial population; otherwise, if the expression of the target gene is low, most of the GFP1-10 will exist in the form of monomers, and the bacterial population will not show obvious fluorescence signal.

[0064] Therefore, by detecting the fluorescence intensity and the bacterial concentration (such as OD600 value) of each transformed strain, and calculating the ratio of the two, the expression efficiency and the soluble level of the target gene in the strains carrying different promoters can be effectively reflected. The higher the expression of the target protein driven by the promoter, the larger the fluorescence / OD600 ratio.

[0065] 3. High-throughput screening process

[0066] a) The above promoter library, target gene-GFP11, and GFP1-10 elements are linked according to the designed cloning sites to construct a complete co-expression plasmid library for high-throughput screening.

[0067] b) The constructed plasmid library is efficiently transformed into the E. coli expression host strain, and the present application preferably uses BL21(DE3).

[0068] c) The transformants are plated on 96-well culture plates and cultured to the logarithmic phase.

[0069] d) Add IPTG to induce gene expression.

[0070] e) After a certain time (e.g. 4 hours) of inducing expression, detect the fluorescence intensity and OD600 value of each well of the bacterial solution using a multifunctional enzyme label meter.

[0071] f) Calculate the fluorescence / OD600 ratio of each transformant strain, and select the strain with the highest ratio (i.e. the best soluble expression level of the target protein).

[0072] g) Perform plasmid sequencing on the selected high-expression strain to identify the type of promoter it carries.

[0073] 4. Optimization of expression strains and applications

[0074] For the recombinant strain selected to carry the best promoter, the soluble expression level of the target protein can be further improved by further optimizing the expression conditions such as IPTG concentration, temperature, induction time, etc.

[0075] This system does not require separate construction and detection of each promoter-gene, and can evaluate the influence of multiple factors simultaneously in a batch and high-throughput manner. It is not only suitable for screening of promoter strength, but also can be extended for evaluation of optimization of RBS, TermiGFP, etc. regulatory elements.

[0076] The present application is simple, efficient and economical to operate, and can be widely applied to optimization of various recombinant protein conditions requiring improved soluble expression, and has important theoretical significance and application prospect in the fields of basic research and biological medicine.

[0077] The raw materials and reagents used in the E. coli promoter screening system and screening method based on isolated green fluorescent protein provided by the present application can be purchased from the market.

[0078] The present application will be further described below in conjunction with examples:

[0079] Example 1

[0080] 1. Construction of target protein and expression plasmid

[0081] a) High-solubility protein: E. coli gmd gene (ACT43810.1)

[0082] The coding sequence of gmd gene was amplified by PCR and fused with GFP11 (16 amino acids, RDHMVLHEYVNAAGIT, as shown in SEQ ID No. 21) coding sequence (as shown in SEQ ID No. 25) at 3' end, with GGGGSGGGGS in between (as shown in SEQ ID No. 22). The amplified product was double digested with EcoRI and BamHI and cloned into the corresponding sites of the above-mentioned pET-28a plasmid vector to construct gmd-GFP11, GFP1-10 expression plasmid. The GFP1-10 coding sequence was amplified by PCR and cloned at the 3' end of gmd-GFP11, expressed by tac promoter.

[0083] b) Low soluble protein: a heterologous superoxide dismutase (SOD) gene

[0084] The SOD gene sequence (WP_010902966.1) was amplified from appropriate biological sources, similar to the gmd gene sequence described above, fused with GFP11 (RDHMVLHEYVNAAGIT, as shown in SEQ ID No. 21) coding sequence at 3' end, and cloned into pET-28a vector to construct SOD-GFP11 expression plasmid. The GFP1-10 (as shown in SEQ ID No. 23) coding sequence (as shown in SEQ ID No. 24) was amplified by PCR and cloned at the 3' end of gmd-GFP11, expressed by tac promoter.

[0085] 2. Transformation and strain culture

[0086] a) Medium preparation

[0087] LB rich medium:

[0088] Bactor Tryptone 10 g / L

[0089] Bactor Yeast Extract 5 g / L

[0090] NaCl 10 g / L

[0091] After high-pressure sterilization of the LB medium, cool it to about 50°C and add kanamycin to a final concentration of 50 ug / ml.

[0092] b) Transform the above-mentioned expression plasmid into E. coli BL21 (DE3) expression host respectively. Use heat shock standard transformation operation.

[0093] c) Pick single colonies from the transformation plate and respectively enlarge culture in kanamycin-resistant LB liquid medium, incubate at 37°C, 220 rpm overnight.

[0094] d) The next day, the overnight culture was scaled up 1:100 into 96-deep well plates containing 1 mL fresh LB medium and incubated until OD 600 of about 0.4.

[0095] 3. Induction of expression and detection

[0096] a) IPTG was added to a final concentration of 0.1 mM and the induction of expression was continued at 30°C, 220 rpm for 4 hours.

[0097] b) The induced bacterial liquid was taken out and the fluorescence intensity and OD 600 values were measured respectively.

[0098] Fluorescence intensity determination:

[0099] 100 μL of the bacterial liquid was taken in a 96-well plate and the fluorescence intensity of each well was determined by a multifunctional enzyme label meter at an excitation wavelength of 485 nm and an emission wavelength of 530 nm (characteristic wavelength of GFP).

[0100] OD600 determination:

[0101] The induced bacterial liquid was slightly diluted so that the OD600 value fell between 0.3 and 0.8, and an appropriate amount was taken into a 1 mL cuvette, and the OD600 value was determined by a spectrophotometer at a wavelength of 600 nm.

[0102] c) The average fluorescence value / OD600 ratio of each sample transformant was calculated in three repeated independent experiments, and the data were analyzed.

[0103] Results:

[0104] Compared with the SOD-GFP11 fusion protein, the fluorescence / OD600 value of the gmd-GFP11 transformant was significantly higher, indicating that the gmd gene derived from E. coli can be expressed more soluble in the host, so that more GFP11 fragments recombine with GFP1-10 in the supernatant to form complete GFP, and the bacterial population emits a stronger fluorescence signal. This index can effectively reflect the level of soluble expression of the target protein. Figure 1 )

[0105] Through the above examples, it can be confirmed that the system of the present application can distinguish different soluble expression proteins, and lay a foundation for subsequent condition optimization screening. If necessary, the specific operation steps of molecular cloning, protein detection, etc. can be carried out according to the conventional molecular biology experimental method.

[0106] Figure 1Figure 2. a) Expression of gmd protein in E. coli. Left side: 25°C culture condition, right side: 37°C culture condition. M: protein molecular weight marker; W: whole cell lysate; "S" and "P" represent supernatant soluble protein and insoluble pellet, respectively. The results show that gmd is expressed efficiently in soluble form at both temperatures. b) Expression of SOD protein in E. coli. Left side: 25°C culture, right side: 37°C culture, labels are the same as a). The results show that SOD is mainly accumulated in insoluble inclusion body form, with low level of soluble expression. c) Detection of soluble expression level of gmd and SOD using the split GFP screening system of the present application. The vertical axis is the ratio of fluorescence intensity / OD600 of the strain, reflecting the soluble expression level of the target protein. The blue column represents gmd expression strain, with a ratio of 185781; the green column represents SOD expression strain, with a ratio of only 37565. The difference between the two is about 5 times, consistent with the above gel electrophoresis results, verifying that the screening system can effectively distinguish target proteins with different solubility.

[0107] Example 2

[0108] 1. Construction of target protein and expression plasmid

[0109] a) Design of iGEM promoter library

[0110] Twenty different strength E. coli synthetic promoter sequences were selected from the iGEM synthetic biology component library, J23100-J23119, the sequences are shown in Table 1:

[0111] Table 1 iGEM promoter sequences

[0112]

[0113]

[0114] The 5' end of each promoter sequence contains an EcoRI restriction site (GAATTC), and the 3' end contains a HindIII restriction site (AAGCTT), which is used to distinguish the restriction sites for subsequent cloning operations.

[0115] b) Target gene P01-GFP11 expression element:

[0116] The gene sequence encoding P01 protein (RDHMVLHEYVNAAGIT, as shown in SEQ ID No. 21) was amplified by PCR, and a GFP11 coding fragment was fused to the 3' end, connected by a GGGGSGGGGS (as shown in SEQ ID No. 22) peptide chain. The amplification product was digested with HindIII and BamHI for later use.

[0117] c) Ptac-driven GFP1-10 expression plasmid:

[0118] Ptac-driven GFP1-10 expression plasmid was constructed according to the method of Example 1.

[0119] d) Construction of single-plasmid co-expression vector:

[0120] The above 20 iGEM promoter sequences were digested with EcoRI / HindIII, P01-GFP11 target gene was digested with HindIII / BamHI, and Ptac-GFP1-10 expression element was digested with EcoRI / BamHI. The three fragments were ligated according to the cloning sites shown in Figure 2 , to construct a co-expression plasmid library of promoter library-P01-GFP11 / Ptac-GFP1-10, with pET-28a(+) as the vector backbone.

[0121] The ligation system was as follows:

[0122]

[0123] The ligation reaction product was transformed into E. coli DH5α competent cells, and single colonies were picked on LB solid plates containing kanamycin to construct the promoter library plasmid library.

[0124] The constructed promoter library plasmid library was efficiently transformed into E. coli BL21(DE3) expression host cells, i.e., the expression strain library for high-throughput screening was obtained.

[0125] 2. Transformation and single colony screening (same as Example 1)

[0126] Single colonies were picked from the transformation plate and inoculated in 96-well plates containing LB liquid medium with kanamycin resistance, and cultured at 37°C, 220 rpm for 4 hours to an OD600 value of 0.4. IPTG was added to a final concentration of 0.1 mM, and the induction expression was continued at 30°C, 220 rpm for 4 hours. 600 The induced bacterial solution was removed, and the fluorescence intensity and OD600 value were measured, respectively. 100 μL of the bacterial solution was taken in a 96-well plate, and the fluorescence intensity of each well was measured using a multifunctional enzyme marker at an excitation wavelength of 485 nm and an emission wavelength of 530 nm (GFP characteristic wavelength). The induced bacterial solution was slightly diluted so that the OD600 value fell between 0.3 and 0.8, and an appropriate amount was taken into a 1 mL cuvette. The OD600 value was measured using a spectrophotometer at a wavelength of 600 nm. Three independent experiments were repeated, the average fluorescence value / OD600 ratio of each sample transformant was calculated, and the data were analyzed.

[0127] 3. Verification of promoter types and comparison of soluble expression

[0128] a) Plasmid sequencing of the three high fluorescence / OD600 ratio strains screened, found that the promoters of the high value strains are all J23118( Figure 3 ) After the strain is optimized by the screening system of the application, the fluorescence / OD600 ratio is increased by about 2.57 times compared with the original T7-P01 strain.

[0129] b) The P01 expression strain driven by the J23118 promoter and the control strain expressing P01 with the original T7 promoter are induced for expression as follows:

[0130] Cultured to OD600 of about 0.6, IPTG was added to a final concentration of 0.1 mM, and induced at 30°C for 4 hours.

[0131] c) Centrifugal collection of bacterial cells, and lysis to collect soluble protein components.

[0132] d) SDS-PAGE electrophoresis is used to compare the soluble expression levels of P01 in the two strains.

[0133] In the gel image, the P01 strain driven by the J23118 promoter will show a significantly stronger soluble protein band, while the band in the original T7 promoter strain is weaker( Figure 4 ). Among them, the left side is the T7-P01 strain driven by the T7 promoter, and the right side is the J23118-P01 strain driven by the J23118 promoter obtained by screening, and each group has three repeats (1-3). Each sample is divided into whole cell lysate ("whole"), supernatant soluble protein ("upper layer") and insoluble precipitate ("precipitate") for SDS-PAGE detection. The results show that in the J23118-P01 strain, the band of P01 protein in the soluble supernatant part is obviously stronger; while in the T7-P01 strain, the protein mainly exists in the insoluble precipitate part, and only has a weak band in the soluble part.

[0134] This confirms that through the screening of the system of the application, an optimized promoter for efficient soluble expression of the target protein can be obtained, and the practical value of the method in optimizing the expression conditions of recombinant proteins is verified.

[0135] The above only describes the preferred embodiments of the application, and it should be noted that for those skilled in the art, without departing from the principles of the application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the application.

Claims

1. A screening system for Escherichia coli promoters based on isolated green fluorescent protein, characterized in that, include: (I) A startup sub-library; the startup sub-library contains multiple startup sequences of different strengths; (II) Target gene-GFP11 fragment, wherein the target gene and the GFP11 fragment are linked by a peptide group; (III) The GFP1-10 fragment driven by the tac promoter; (IV) Plasmid vector, wherein the plasmid vector contains multiple restriction sites for cloning the promoter library, the target gene-GFP11 fragment and the GFP1-10 fragment.

2. The E. coli promoter screening system as described in claim 1, characterized in that, The promoter library consists of 20 E. coli promoters of different strengths from iGEM, each with EcoRI and HindIII restriction sites attached upstream and downstream, respectively.

3. The E. coli promoter screening system as described in claim 1 or 2, characterized in that, The peptide group has: (I) The amino acid sequence as shown in SEQ ID No. 22; (II) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and which has the same function as the amino acid sequence described in (I); or (III) An amino acid sequence having 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or more of the same identity as the amino acid sequence described in (I) or (II).

4. The *E. coli* promoter screening system according to any one of claims 1 to 3, characterized in that, The upstream of the target gene-GFP11 fragment is a HindIII restriction site, and the downstream is a BamHI restriction site; and / or The upstream of the GFP1-10 fragment is an EcoRI restriction site, and the downstream is a BamHI restriction site. The single plasmid vector includes pET-28a(+).

5. The host, characterized in that, The transformation is performed using the Escherichia coli promoter screening system as described in any one of claims 1 to 4.

6. The host as described in claim 5, characterized in that, It includes Escherichia coli; Preferably, the strain of Escherichia coli includes BL21(DE3).

7. The application of any of the following items in screening promoters; (I) The E. coli promoter screening system as described in any one of claims 1 to 4; or (II) The host as described in claim 5 or 6.

8. A method for screening Escherichia coli promoters using the Escherichia coli promoter screening system as described in any one of claims 1 to 4, characterized in that, Includes the following steps: a) Construct the promoter library, the target gene-GFP11 fragment, and the GFP1-10 fragment; clone them into the plasmid vector; b) Transform the constructed plasmid vector into an Escherichia coli expression host strain; c) Inducing expression of the target gene-GFP11 and GFP1-10 fragments; d) Measure the fluorescence intensity and cell concentration of each transformant; e) Calculate the ratio of the fluorescence intensity to the bacterial cell concentration, and screen out the transformants with the highest ratio; f) Sequencing the transformant with the highest ratio to identify the promoter sequence it carries.

9. The method as described in claim 8, characterized in that, The induction in step c) is performed using IPTG induction.

10. A computer device, computer-readable storage medium, or computer program product, characterized in that, The computer device includes a memory and a processor, the memory storing a computer program, and the processor executing the computer program to implement the steps of the method as described in claim 8 or 9; or The computer-readable storage medium stores a computer program that, when executed by a processor, implements the steps of the method as described in claim 8 or 9; or The computer program product includes a computer program that, when executed by a processor, implements the steps of the method as described in claim 8 or 9.

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