Quantitative characterization method for genotype phenotype based on transposon mutation and high-throughput sequencing

Abstract

The invention relates to the field of genomics, and discloses a genotype phenotype quantitative characterization method based on transposon mutation and high-throughput sequencing, which comprises the following steps: designing and constructing a recording transposon and a temperature-sensitive transposon plasmid; the method comprises the following steps: introducing a temperature-sensitive transposon plasmid into a host strain to obtain a transposon strain, inducing a recording transposon to be inserted into a host genome through temperature regulation and control, carrying out plasmid loss, carrying out amplification culture, and carrying out verification and evaluation to obtain a transposon mutant library which is verified to be qualified; and inoculating the transposon mutant library to a culture system corresponding to the target phenotype. The intracellular dynamic phenotypic difference is converted into a stable sequenced fusion transcript signal through fusion design of a recording type transposon and a phenotypic response type biosensor, construction of a mutant library mediated by temperature-sensitive transposon plasmids, collaborative sequencing of double sequencing libraries and correction of quantitative errors by a unique molecular identifier.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of genomics, in particular to a genotype phenotype quantitative characterization method based on transposon mutation and high-throughput sequencing. BACKGROUND

[0002] After the completion of the Human Genome Project and the sequencing of the genomes of various organisms, functional genomics has become a research hotspot. However, traditional gene knockout, RNA interference and other methods are difficult to realize genome-wide gene-phenotype correlation analysis due to their complexity and high cost. With the development of high-throughput sequencing technology, Tn-seq technology was introduced around 2009. It can analyze gene function in high throughput by constructing a mutation library through random insertion of transposons and locating the insertion sites through sequencing. It has also derived improved technologies such as HITS, TraDIS and CRISPR-Tn-seq. On this basis, Sort-Seq technology introduces flow cytometry sorting and fluorescent reporter genes, and gets rid of the dependence on survival screening of traditional Tn-seq, which is suitable for gene regulation and metabolic pathway optimization research.

[0003] However, Sort-Seq has obvious technical limitations: first, it is highly dependent on expensive flow cytometry sorting equipment, which is complex to operate and has limited popularization; second, sorting is carried out under non-physiological conditions (such as buffer, low temperature), which can easily change the cell state and interfere with the results; third, it cannot exclude the influence of cell growth rate difference, and the growth advantage can mask the real phenotype; fourth, the sorting accuracy depends on the fluorescent signal, and the phenotype sorting effect is poor for weak or uneven signals. SUMMARY

[0004] In view of the deficiencies of the prior art, the present application provides a genotype phenotype quantitative characterization method based on transposon mutation and high-throughput sequencing, which solves the problems of low throughput, high cost, dependence on large sorting equipment and inability to effectively exclude growth difference interference in the existing functional genomics research.

[0005] To achieve the above purpose, the present application realizes the following technical scheme: a genotype phenotype quantitative characterization method based on transposon mutation and high-throughput sequencing, comprising the following steps: Design and construct a recording-type transposon and a temperature-sensitive transposon plasmid; Introduce the temperature-sensitive transposon plasmid into a host strain to obtain a transposition strain, induce the recording-type transposon to insert into the host genome and perform plasmid loss through temperature regulation, verify and evaluate after expansion culture, and obtain a verified transposon mutant library; Inoculate the transposon mutant library into a culture system corresponding to the target phenotype, so that the genotype difference caused by the insertion of the recording-type transposon is converted into a phenotype difference, the phenotype difference is converted into a sequencable fusion transcript through the biological sensor of the recording-type transposon, and the bacterial cells are collected after the culture is completed; extracting total DNA and total RNA of the bacterial bodies, and constructing a genomic DNA library for genotyping and an RNA-cDNA library for quantitative phenotyping, respectively; sequencing the genomic DNA library and the RNA-cDNA library, and performing bioinformatics analysis to locate the insertion site of the recording transposon, quantify the fusion transcript, and correlate the genotype and the phenotype, so as to screen key candidate genes for regulating the target phenotype; performing scarless knockout on the key candidate genes, and verifying the regulation function of the genes on the target phenotype through fermentation experiments and phenotype detection.

[0006] By adopting the technical solution, the intracellular dynamic phenotype difference is converted into a stable and sequencable fusion transcript signal through the fusion design of the recording transposon and the phenotype response type biosensor, the mutant library construction mediated by the temperature-sensitive transposition plasmid, the collaborative sequencing of the double sequencing libraries, and the correction of the quantitative error by the unique molecular identifier, the location of the transposon insertion site and the counting of the abundance of the fusion transcript are realized, the quantitative deviation caused by the interference of non-physiological condition sorting on the cell state and the difference in cell growth rate is avoided, the high-throughput, high-sensitivity and high-precision quantitative correlation analysis of the massive microbial mutant genotypes and phenotypes is realized, and the problems of low throughput, high cost, dependence on large-scale sorting equipment and inability to effectively exclude the growth difference interference in the existing functional genomics research are solved.

[0007] Preferably, the recording transposon comprises a transposition mediation unit, a screening marker unit and a phenotype recording unit. The transposition mediation unit is the Mariner himar1 C9 transposase-specific inverted repeat sequence at the left and right ends of the recording transposon, the Mariner himar1 C9 transposase is a variant optimized by directional mutation, and only the 5'-TA-3' sequence is inserted into the host genome. The screening marker unit is the spectinomycin resistance gene in the middle region of the recording transposon. The phenotype recording unit is an integrated phenotype response type biosensor, the biosensor is composed of a regulatory protein coding gene and a reporter gene, and no transcription terminator is arranged downstream of the reporter gene, so that the transcription can be read-through from the biosensor promoter to the host genome downstream of the transposon insertion site, forming a sensor-host genome fusion transcript.

[0008] Preferably, the temperature-sensitive transposition plasmid takes a plasmid containing a temperature-sensitive replicon as a backbone, which is used to carry the recording transposon and perform subsequent plasmid loss.

[0009] Preferably, the construction of the transposon mutant library specifically comprises the following steps: Transposon strain preparation: the temperature-sensitive transposon plasmid was introduced into the host strain by electroporation, and after electroporation, the LB solid plate containing ampicillin was coated and cultured at 30℃ for 16h. The single colony was picked to obtain the transposon strain; Transposition induction: the single colony of the transposon strain was inoculated into the LB liquid medium containing ampicillin, and the seed liquid was obtained by overnight culture. The seed liquid was transferred to the LB liquid medium containing spectinomycin and IPTG, and was cultured at 16℃ overnight to induce the expression of transposase, and the bacterial liquid was obtained; Plasmid loss and library expansion: the bacterial liquid was transferred to the antibiotic-free LB liquid medium and cultured overnight to lose the plasmid, and then was transferred to the LB liquid medium containing spectinomycin and cultured overnight to expand the library; Library preservation: the bacterial liquid after expansion culture was added with 50% glycerol at a volume ratio of 1:1, and was stored at -80℃ to obtain the transposon mutant library.

[0010] Preferably, the verification evaluation specifically includes: Insertion randomness verification: Tail PCR method is used, the genomic DNA of the transposon mutant library is used as the template, transposon-specific nested primers and random degenerate primers are used for three rounds of nested PCR, and the PCR product is sequenced and aligned to the host genome to verify randomness; Whole genome coverage verification: DNA footprinting method is used, specific primer pairs of essential genes, non-essential genes and random genes are designed, and the coverage of the transposon on the whole genome is verified by the distribution of PCR product bands.

[0011] Preferably, the phenotype-responsive biosensor is used to perceive the intensity of intracellular specific molecular signals, and when the target phenotype is tryptophan metabolism phenotype, the phenotype-responsive biosensor is composed of tnaC a gene fused with an enhanced green fluorescent protein gene.

[0012] Preferably, the construction of the genomic DNA library includes the following steps: The total DNA extracted is fragmented, end-repaired and dA-tailed to form dA-tailed DNA fragments; The dA-tailed DNA fragments are added with a specially designed UMI (Unique Molecular Identifier) adapter containing a unique molecular identifier and a ligase for adapter ligation reaction, and the ligation product is obtained by magnetic bead purification after the reaction is completed; The ligation product is used as the template, and the transposon-specific primers and sequencing adapter primers are used for Nest PCR to enrich the Nest PCR amplification product containing the transposon flanking sequence; The Nest PCR product is used as a template, and an Index primer with a sample distinguishing tag is added to perform Index PCR, and after the reaction is completed, the genomic DNA library is obtained by magnetic bead purification.

[0013] Preferably, when constructing the RNA-cDNA library, a unique molecular identifier is introduced in the reverse transcription stage, which is used to remove PCR amplification bias in data analysis and count the original molecular number of fusion transcripts.

[0014] Preferably, the high-throughput sequencing adopts the Illumina NovaSeq 6000 platform for double-end sequencing, and the bioinformatics analysis includes: using BWA software to align the sequencing data of the genomic DNA library to the host reference genome to locate the transposon insertion site, using DESeq2 software to perform differential analysis on the quantitative data of the fusion transcript, and screening to obtain key candidate genes regulating the target phenotype.

[0015] Preferably, the scarless knockout adopts the CRISPR / Cas9 technology, the specific gRNA of the key candidate gene is cloned into the pCas9 vector and then introduced into the host strain, and the Cas9 protein expression is induced to realize gene knockout; the phenotype detection adopts the ultra-high-throughput mass spectrometer, and the content of the target substance in the fermentation product is detected to verify the regulation function of the knockout gene on the target phenotype.

[0016] The application provides a genotype phenotype quantitative characterization method based on transposon mutation and high-throughput sequencing. 1、The application converts the intracellular dynamic phenotype difference into stable and sequencable fusion transcript signals by the fusion design of recording type transposon and phenotype responsive biosensor, the mutant library construction mediated by temperature sensitive transposon plasmid, the cooperative sequencing of double sequencing libraries, and the correction of quantitative error by unique molecular identifier, realizes the positioning of transposon insertion site and the counting of fusion transcript abundance, avoids the quantitative deviation caused by the interference of non-physiological condition sorting on cell state and the difference of cell growth rate, and realizes the high-throughput, high sensitivity and high precision quantitative correlation analysis of massive microbial mutant genotypes and phenotypes.

[0017] 2、The present application converts intracellular phenotype signals directly into sequencable fusion transcripts by integrating phenotype-responsive biosensors in transposons, without relying on expensive flow cytometry sorting equipment required by existing Sort-Seq technology, by completing phenotype "recording" under normal physiological conditions of microbial growth and fermentation, ensuring that cells are in a natural metabolic state, avoiding interference of non-physiological environment on phenotype signals, by introducing unique molecular identifiers during RNA-cDNA library construction, correcting quantitative errors caused by PCR amplification bias and cell growth rate differences, realizing accurate counting of the original number of fusion transcripts, and guaranteeing the authenticity and reliability of genotype-phenotype correlation results.

[0018] 3、The present application can analyze hundreds of thousands to millions of independent mutants in parallel by relying on double-library high-throughput sequencing technology, breaking through the throughput limit of existing flow sorting technology, realizing genotype-phenotype correlation screening in the whole genome range, and at the same time, phenotype signals accumulate in the form of fusion transcripts in cells, even if the intracellular low concentration of metabolites or weak signal regulation of phenotype difference, can be captured by the depth coverage of high-throughput sequencing and the accurate counting of UMI, which can effectively analyze the functional genes corresponding to weak phenotype differences, and expand the coverage range of functional genomics research.

[0019] 4、The present application can quickly adapt to the quantitative characterization requirements of different phenotypes by replacing the response elements of biosensors, without the need for overall modification of the technology system, and without relying on the genetic background of specific host strains, and can be applied to Escherichia coli, yeast, actinomycetes and other microorganisms, providing a universal tool for functional genomics research and metabolic engineering of different microbial systems. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a schematic diagram of the basic principle of the present application; Figure 2 is a comparison diagram of the principle of traditional Tn-seq technology (A) and the principle of Tn-Rec-Seq proposed by the present application (B); Figure 3 is a schematic diagram of Mariner himar1 C9 transposon of the present application; Figure 4 is a schematic diagram of the transposon plasmid of the present application; Figure 5 is a temperature-sensitive plasmid loss verification diagram of the present application; Figure 6 is a Tail PCR agarose gel electrophoresis diagram of the present application; Figure 7 is a mutant library Foot-printing essential gene and non-essential gene insertion verification diagram of the present application; Figure 8Verification chart of random gene insertion for the mutant library footprinting of the application; Figure 9 Schematic diagram of tryptophan response transposon plasmid of the application; Figure 10 Flow chart of data analysis of the application; Figure 11 Intracellular and extracellular tryptophan content chart of the single knock-out strain of the application. DETAILED DESCRIPTION

[0021] The technical solutions of the application will be clearly and completely described below with reference to the drawings of the application. Obviously, the described embodiments are only a part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.

[0022] Technical concept: The application aims to solve the problems of low throughput, high cost, dependence on large sorting equipment and inability to effectively exclude growth difference interference in genotype and phenotype correlation analysis in existing functional genomics research. The application provides a genotype and phenotype quantitative characterization method based on transposon mutation and high-throughput sequencing, which can quantitatively characterize the correlation between a large number of mutant genotypes and phenotypes with high throughput and high sensitivity. A technology system named “Transposon-Recorder-Sequencing (Tn-Rec-Seq)” is established. It is divided into two core parts: 1) high-throughput genotype construction module; 2) high-throughput genotype characterization module.

[0023] 1) High-throughput genotype construction module: transposon random insertion technology is used to construct a large-scale and diverse mutant library in the host cell genome.

[0024] 2) High-throughput genotype characterization module: a phenotype response type biosensor integrated in the transposon is designed. The sensor can sense the intensity of intracellular specific molecular signals (such as metabolite concentration) and convert it into a sequencable transcription output signal. By high-throughput sequencing of specific DNA fragments (locating genotypes) and fused mRNA fragments (quantifying phenotypes), the accurate quantitative correlation between genotypes and phenotypes is finally realized.

[0025] Please refer to the drawings of the application Figure 1 - the drawings of the application Figure 11 The purpose of the application is to overcome the problems of low throughput, high cost, dependence on large sorting equipment and inability to effectively exclude growth difference interference in genotype and phenotype correlation analysis in existing functional genomics research, and to create a new technical method, the core goal of which is: By transcribing phenotypic signals at the transcriptional level of sequenceable mRNA within cells, interference and bias caused by physical sorting can be avoided.

[0026] By organically integrating transposon mutation technology, transcriptional regulatory biosensors, and transcriptome sequencing, we can directly capture and accurately locate the phenotypic responses of microbial mutants, overcoming the limitations of traditional Tn-Seq in terms of phenotypic resolution, functional association accuracy, and sequencing data integration.

[0027] By introducing molecular tags (UMI) and other methods, quantitative errors caused by differences in cell growth rate and PCR amplification bias can be effectively eliminated, achieving more accurate quantitative analysis.

[0028] Reduce reliance on flow cytometry cell sorting equipment, thereby reducing experimental costs and complexity.

[0029] Establish a genotype-phenotype high-throughput association analysis platform that is more universal, more sensitive, and easier to operate.

[0030] This invention proposes Tn-Rec-Seq (Transposon-Recorder-Sequencing) technology, which integrates transposon random insertion, phenotypic response biosensor design, and transcriptome sequencing. This enables the direct capture and high-precision localization of phenotypic responses in microbial mutants, overcoming the limitations of traditional Tn-Seq in phenotypic resolution, functional association accuracy, and sequencing data integration. This technology is particularly suitable for functional gene screening based on metabolic flux change phenotypes (such as tryptophan production), providing a new research tool for metabolic engineering and synthetic biology.

[0031] The Tn-Rec-Seq technology proposed in this invention integrates a "phenotypic recorder" (i.e., biosensor) into a transposon, enabling the direct conversion of dynamic phenotypic information into stable, accumulative mRNA molecular signals within cells, which are then decoded via high-throughput sequencing. Figure 1 As shown, taking the screening of genes that affect the concentration of intracellular metabolites as an example, the specific implementation plan includes the following five steps: Step 1: Core Component Design and Construction – Construction of the “Recording Type” Transponder Design and construct a special transposon. Besides containing the necessary transposable components, the core of this transposon integrates a phenotypic responsive biosensor module. This module includes: A regulatory protein that responds to a specific intracellular signal (such as tryptophan concentration) and its corresponding promoter.

[0032] Downstream of this promoter, a reporter gene sequence that can be sequenced is placed.

[0033] Key design: No transcriptional terminator is placed downstream of the reporter gene. This design allows the transcription to start from the sensor promoter, "read-through" into the host genome region downstream of the transposon insertion site, forming a "sensor-host genome" fusion transcript. The abundance of this fusion transcript directly reflects the intracellular signal strength.

[0034] Step 2: Construction of high-throughput mutant library The "recorder" transposon constructed above is introduced into host cells (e.g. E. coli), and through the action of transposase, the transposon is randomly inserted into different positions of the host genome, constructing a diversity library containing hundreds of thousands or even millions of independent mutants.

[0035] Step 3: "Recording" process of phenotype-genotype correlation The mutant library is cultured under specific culture conditions. In each cell: The transposon insertion leads to a change in the function of a certain gene (e.g. inactivation, enhancement or attenuation).

[0036] The change in gene function can cause a change in the concentration of intracellular target metabolites (e.g. tryptophan).

[0037] The change in metabolite concentration is sensed by the biosensor on the transposon, which regulates the transcriptional activity of its promoter.

[0038] The level of transcriptional activity determines the amount of fusion transcript generated. Therefore, the abundance of the fusion transcript becomes the "molecular record" of the phenotype strength of the cell.

[0039] Step 4: Construction of sequencing library and high-throughput sequencing After the culture ends, all cells are collected, and total DNA and total RNA are extracted. Two types of sequencing libraries are constructed: DNA library (for genotype mapping): The connection region between the transposon and the flanking genomic DNA is amplified by specific primers, which is used to accurately locate the insertion site of each transposon in the genome.

[0040] RNA-cDNA library (for phenotype quantification): First, total RNA is reverse transcribed into cDNA. Then, using specific primers targeting the sensor reporter gene region, the aforementioned fusion transcript cDNA is enriched and amplified. The read count of each sequence in this library reflects the phenotype strength of the corresponding mutant.

[0041] Introduction of UMI: A unique molecular tag (UMI) is added to each initial DNA / RNA molecule during the reverse transcription or adapter ligation step of library construction. In subsequent data analysis, UMI can be used to remove quantitative bias caused by PCR amplification preference, realize the absolute count of the original molecule number, and greatly improve the accuracy of quantification.

[0042] Step 5: Bioinformatics analysis and functional gene mining After high-throughput sequencing of the two libraries, data analysis is performed: Genotype analysis: Align the sequencing data of the DNA library to the reference genome to obtain the accurate map of all transposon insertion sites in the whole library.

[0043] Phenotype quantification: Analyze the RNA-cDNA library data, use UMI to correct reads, and obtain the accurate expression of each insertion site corresponding to the fusion transcript.

[0044] Correlation analysis: Correlate the genotype map with the phenotype quantification data. Through statistical analysis, filter out those genes whose insertion will cause the expression of fusion transcripts to increase or decrease significantly. These genes are the key candidate genes for regulating the target phenotype (such as tryptophan synthesis, degradation, and transport). As shown in Figure 2 The principle of traditional Tn-seq technology (A) and the principle of Tn-Rec-Seq proposed by the present technology (B) are shown in the following figure (Note: Sensor gene can also be expressed independently on intracellular plasmid or genomic DNA).

[0045] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0046] The data were processed using Graphpad prism 9.0 statistical software, and the experimental results were expressed as mean values.

[0047] The strain is Escherichia coli K-12 MG1655 ΔendA ΔrecA (DE3) The culture medium for culturing the strain is LB medium, and each liter of LB medium contains: 20 g of proteose peptone, 5 g of yeast extract, 20 g of sodium chloride, and 20 g of agar powder (added to LB solid medium). The resistance medium is LB liquid or solid medium added with 50 μg / mL of spectinomycin hydrochloride or 100 μg / mL of ampicillin.

[0048] The tryptophan shake flask fermentation liquid medium is used for verifying the tryptophan yield of the strain, and the tryptophan fermentation liquid medium contains 20 g of glucose, 7.5 g of dipotassium hydrogen phosphate trihydrate, 2.0 g of magnesium sulfate heptahydrate, 1.6 g of ammonium sulfate, 0.0756 g of ferrous sulfate heptahydrate, and 1% (v / v) of 1000x trace element mother liquor per liter of volume, wherein the 1000x trace element mother liquor contains 4.5 g of manganese sulfate monohydrate, 20 g of sodium sulfate, 6.4 g of zinc sulfate heptahydrate, 4.0 g of cobalt sulfate hexahydrate, and 0.6 g of copper sulfate pentahydrate per liter of volume.

[0049] Example 1: High-throughput random genotype construction based on transposon mutation 1. Design and construction of transposon structure plasmid: The present application uses Mariner himar1 C9 transposase transposase for subsequent construction of transposon mutation library, Mariner Himar1 C9 transposase is a transposase variant derived from insects, belonging to the mariner / Tc1 transposon family, which is widely used in transposon sequencing experiments, and Himar1 C9 is a variant optimized by directional mutation of Himar1, including C134S and 8 additional amino acid changes, which shows higher activity in vitro or heterologous system, the transposon mainly inserts into 5'-TA-3' sequence, the sequence bias is relatively low, only the TA base pair in the center of the target sequence can realize DNA bending, and Himar1 has high activity in bacteria. The transposon structure is shown in Figure 3 The left and right ends are specific recognition sequences of Mariner himar1 C9 transposase (red), and the middle part of the transposon is a spectinomycin antibiotic resistance gene, as shown in the accompanying Figure 3 .

[0050] 2. Construction of plasmid that can be lost: In order to easily obtain the transposon mutation library, the inventors use the method of introducing plasmid into host strain to obtain the transposon mutation library, and at the same time, in order to prevent the transposon plasmid from affecting the growth of the strain and affecting the subsequent library sequencing data analysis, the inventors use a temperature-sensitive RepA-dependent replicon when constructing the plasmid. The replicon is stably replicated at 30~34℃, and when the culture temperature is greater than or equal to 37℃, the RepA protein structure is unstable or inactivated, and the plasmid cannot be normally replicated, so according to the characteristics of this temperature-sensitive replicon, the transposon plasmid can be successfully lost by changing the culture temperature. The constructed temperature-sensitive transposon plasmid is shown in the accompanying Figure 4 , which can be successfully lost after 3 days of culture at 42℃, as shown in the accompanying Figure 5 .

[0051] 3. Construction of transposition strains: The temperature-sensitive transposition plasmid was transformed into the host strain by electroporation. Escherichia coli K-12 MG1655 Δ endA ΔrecA The (DE3) strain was obtained Escherichia coli K-12 MG1655 ΔendA ΔrecA (DE3)-1 transposition strain.

[0052] 4. Construction of transposon mutant library: After obtaining the transposition strain, the construction of the transposon mutant library was carried out. The construction process is as follows: (1) Select positive transformants and inoculate in 5 mL LB (containing Amp 100 μg / mL) at 30 ℃, 200 rpm / min overnight culture of positive transformants.

[0053] (2) Take 200 μL of bacterial solution and add to 5 mL LB liquid medium containing Spec (50 μg / mL) and IPTG (1 mM IPTG), 16 ℃, 200 rpm / min, overnight culture, while taking an appropriate amount of bacterial solution for gradient dilution, taking 10 μL respectively on no resistance LB plate, only Amp-containing LB plate and only Spc-containing LB plate at 30 ℃ after culture for counting.

[0054] (3) Add 200 μL of the culture to 200 mL of non-antibiotic LB liquid medium preheated at 42 ℃, and incubate at 42 ℃, 150 rpm / min overnight. Count the colonies on the three culture plates after incubation at 42 ℃.

[0055] (4) Add 2 mL of the culture to 200 mL of LB liquid medium preheated at 42 ℃, and incubate at 37 ℃, 150 rpm / min overnight. Count the colonies on the three culture plates after incubation at 42 ℃ (three replicates).

[0056] (5) Add 2 mL of the culture to 200 mL of LB liquid medium containing Spc (50 μg / mL) preheated at 42 ℃, and incubate at 42 ℃, 150 rpm / min overnight. Count the colonies on the three culture plates after incubation at 42 ℃ (three replicates). (6) Add several mutant libraries to 50% glycerol and store at -80 ℃.

[0057] Preliminary quality assessment of transposon mutant library The randomness of transposon insertion position in the constructed library was verified using Tail PCR method, and the whole gene coverage of transposon was verified using DNA footprinting method. The specific conditions of the two methods are as follows: (1) Tail-PCR verification of transposon insertion randomness The single colony of transposon insertion mutant bacteria was picked and inoculated in 1 mL of LB culture solution containing 100 μg / mL ampicillin or 50 μg / mL spectinomycin, and cultured overnight at 37°C on a shaker; Genomic extraction; Three nested primers SP1, SP2 and SP3 were designed according to known sequences as long primers, and a random degenerate primer AD was used as a short primer. Three rounds of PCR reactions were performed using the primers PF and AD as a pair and total bacterial DNA as a template. The primer sequences are shown in Table 1, the reaction system is shown in Table 2, and the reaction program is shown in Table 3.

[0058] Table 1 Primers used in Tail PCR

[0059] Table 2 Reaction system of Tail PCR

[0060] Table 3 Reaction program of Tail PCR

[0061] After agarose gel electrophoresis detection, the company used primer SP3 for first-generation sequencing, and the agarose gel electrophoresis results are shown in FIG. 1. The clearest band at the top of the lane was cut and sent for measurement. The sequencing results were compared with NCBI using Blast, and the comparison results are shown in Table 4. The sequencing results show that the transposon is randomly inserted into the genome. Figure 6 Table 4 Sequencing results of Tail PCR

[0062] DNA footprinting verifies the whole genome coverage of transposon insertion

[0063] The whole genome coverage of transposon insertion in the mutant library was evaluated by DNA footprinting. First, a downstream primer was designed based on the transposon sequence, and an upstream primer was designed on three different types of target genes in the E. coli genome. The three types of target genes are essential genes (rpoB), non-essential genes (lacZ), and genes with unknown functions (yddG). ftsZ, rplA, yigP, murA, fabZ aroF, cadA, dacB, hlyE, hsdS, maeB, pykF, tyrB, waaG, yihI ​​The study included ten random genes (randomly selected from the genomic backbone). Total genomic DNA was extracted from the mutation library and used as a template. A series of products were amplified using the primers described above, each corresponding to a transposon insertion mutant from the mutation library. For essential genes, transposon insertion into these genes leads to strain death, and PCR amplification typically produces large bands without many bands of varying sizes. However, when transposons are inserted into non-essential or random genes, many bands of different sizes are formed. (See attached image.) Figure 7 and attached Figure 8 As shown, no bands of varying sizes appeared after amplification with primers for the four essential genes, while bands of varying sizes were produced after amplification with primers for non-essential genes and random genes. These results indicate that the mutant library transposons are randomly inserted and have a high insertion coverage across the entire E. coli genome.

[0064] Example 2: Construction and Phenotypic Analysis of Random Transposon Mutant Genotype Library Based on NGS Sequencing In this embodiment, tryptophan, which can be produced endogenously in E. coli, was selected as phenotypic information to construct a tryptophan-responsive transposon mutant library. The tryptophan response used a tryptophan intracellular sensor reported in the literature, and the constructed plasmid is shown below. Figure 7 As shown. This biosensor is made of tnaC Composition, its encoding tnaCAB The operon's leader sequence (whose expression is induced by L-Trp) is fused downstream to an enhanced green fluorescent protein (EGFP) gene. The response mechanism of this tryptophan biosensor is as follows: tnaCAB The enzyme encoded by the operon is involved in the catabolism of tryptophan, and its expression is regulated by tryptophan concentration. Specifically, tnaC yes tnaCAB The leader peptide of the operon is expressed induction by tryptophan. In the absence of tryptophan, tnaC The leader peptide is released from tRNA and ribosomes, allowing the Rho factor to access... tnaCAB The Rho-dependent terminator on the transcription factor prematurely terminates transcription. In the presence of tryptophan, the contact of the Rho factor is blocked. tnaCAB Transcription continues. In constructing the L-Trp sensor, tnaC The coding sequence of [protein name] is fused with the coding sequence of enhanced green fluorescent protein (EFPP) to form a fusion protein. Thus, when the intracellular tryptophan concentration changes, [the protein's] coding sequence is fused with the coding sequence of EFPP, forming a fusion protein. tnaC The expression level of tryptophan also changes, thus affecting EGFP expression. By monitoring the fluorescence signal of EGFP, the concentration of tryptophan in cells can be indirectly monitored. When the concentration of tryptophan increases, tnaCIncreased expression of tryptophan leads to increased EGFP expression and enhanced fluorescence signal; conversely, decreased tryptophan concentration results in weakened fluorescence signal. Therefore, by detecting the intensity of the fluorescence signal, the intracellular tryptophan concentration can be monitored in real time.

[0065] Fluorescence signals can be used to screen strains with increased tryptophan production using flow cytometry, but this method is indirect, and the equipment is expensive and has limited throughput. Therefore, this invention proposes recording intracellular tryptophan phenotype by recording response signals at the RNA level. The specific principle is to record the response signals at the RNA level... Figure 9 As shown, tryptophan-responsive transposon plasmids randomly insert transposons containing tryptophan sensors into the E. coli genome. When the sensor responds to intracellular tryptophan, the transcriptional levels of genes containing GFP and downstream of GFP inserted into the genome increase. After constructing specialized transcriptome sequencing libraries and genome libraries, next-generation sequencing is performed. The obtained data is then subjected to in-depth data analysis to obtain the location of the tryptophan-responsive transposon inserted into each gene or intergenetic region, as well as transcriptome data showing different transcriptional levels due to different tryptophan production. This allows for high-throughput, high-speed, and inexpensive association between phenotypes and genotypes at the RNA level.

[0066] Example 3: Construction of sequencing libraries for phenotypic information response transposon mutant libraries Transcriptome-based library construction protocol: PCR amplification is unavoidable during library construction, inevitably introducing PCR amplification bias that can lead to errors in data analysis. Therefore, this invention introduces UMI (Unique Memory Injection) before PCR amplification during library construction. This allows UMI counting analysis to eliminate bias regardless of the number of amplification cycles, as detailed below: (1) Purification with Poly(A) and magnetic beads: Kit: E. coli Poly(A) Polymerase (NEBM0276S), as follows:

[0067] 1) Incubate the above system at 37°C for 2 minutes.

[0068] 2) Add 5 µL of 50 mM EDTA to stop the reaction. The final volume is 25 µL.

[0069] 3) Add 45 µL of RNA clean beads to the reaction.

[0070] 4) Incubate at room temperature for 5 minutes.

[0071] 5) Centrifuge the sample and place the precipitate on a magnetic rack. Keep the tube on the magnet, remove and discard the supernatant.

[0072] 6) Keep the tube on the magnet and wash the beads with 200 µL freshly prepared 70% ethanol. Carefully invert the tube on the rack 180° twice to wash the pelleted beads. Aspirate and discard the supernatant.

[0073] 7) Repeat the above steps for a total of two washes.

[0074] 8) After a brief spin, place the tube back on the magnet. Aspirate any residual ethanol. Allow it to dry for 30 seconds, but do not dry the pellet to the point of cracking.

[0075] 9) Remove the tube from the magnetic rack and resuspend the pellet in 12 µL nuclease-free water. Incubate on ice for 5 minutes.

[0076] 10) Pellet the beads on the magnet until the eluate is clear and colorless.

[0077] 11) In a clean 1.5 mL microcentrifuge tube, remove and retain 12 µL of the eluate, which contains the 3'-polyadenylated RNA.

[0078] 12) Remove 1 µL of the final eluate and quantify the final concentration using the BR RNA Qubit kit.

[0079] 13) Store the final product at -80°C, or proceed immediately to library preparation, keeping the sample on ice.

[0080] (2) cDNA single strand synthesis Preparation: kit: Hiscript 1st Strand cDNA synthesis kit (+gDNA wiper) (Vazyme R412) Operation procedure: 1) RNA template denaturation

[0081] Heat the above system at 65°C in a PCR instrument for 5 min, quickly place on ice for quenching, and stand on ice for 2 min 2) Genomic DNA removal Add 2 µL 5×gDNA wiper Mix to the above system, mix gently with a pipette. Place in a preheated PCR instrument at 42°C for 2 min.

[0082] 3) Prepare the first strand cDNA synthesis reaction solution Add to the above system: 5 µL 4×HiScript IV RT SuperMix 3 μL 1 μM P7-short-BUMI-T 2 μL RNase-free ddH2O Mix gently with pipette 4) Perform first strand cDNA synthesis reaction under the following conditions: 50℃ 30 min; 85℃ 5 s (3) PCR and product purification Reagent: Yousan 2x Hieff Canace plus PCR Master Mix (with Dye) (10154) 1) Perform PCR Each system is as follows:

[0083] Reaction program:

[0084] 2) PCR product purification Combine the PCR products of the above systems in a 2 mL EP tube; After combining, use 1.2 times the volume of magnetic beads for purification; The purification steps are as follows: After the magnetic beads are equilibrated to room temperature, invert or vortex to mix VAHTS DNA Clean Beads; Take 1.2 times the volume of magnetic beads DNA Clean Beads into the sample system, vortex slightly or use Gently pipette 10 times to mix thoroughly; Incubate at room temperature for 5 min; Centrifuge the 2 mL EP tube briefly and place it on the magnetic stand. After the solution is clear (about 5 min), carefully remove the supernatant; Keep the 2 mL EP tube on the magnetic stand at all times, slowly add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads, incubate at room temperature, and carefully remove the supernatant; Repeat step 5, a total of two rinses; Keep the 2 mL EP tube on the magnetic stand at all times, air dry the magnetic beads for 3-5 min without ethanol residue; Remove the 2 mL EP tube from the magnetic stand and elute.

[0085] (4) Index PCR Reagent: Yousan 2x Hieff Canace plus PCR Master Mix (with Dye) (10154) The system is as follows:

[0086] Reaction procedure:

[0087] (5) PCR product purification 0.9 times the volume of magnetic beads were used for purification, and the purified product was eluted using 22 μL of RNase-free ddH2O. The final product was 20 μL for sequencing, and 1 μL for HS Qubit kit to determine the concentration.

[0088] After the magnetic beads were equilibrated to room temperature, the VAHTS DNA Clean Beads were mixed by inverting or vortexing; 0.9 times the volume of DNA Clean Beads was aspirated into the sample system, and mixed well by vortexing or gently pipetting 10 times with a pipette; Incubate at room temperature for 5 min; Centrifuge the 1.5 mL EP tube briefly and place it on the magnetic stand. After the solution is clear, carefully remove the supernatant; Keep the 1.5 mL EP tube on the magnetic stand at all times, slowly add 200 μL of freshly prepared 70% ethanol (equilibrated to room temperature) to rinse the magnetic beads, incubate at room temperature, and carefully remove the supernatant; Repeat step 5 for a total of two rinses; centrifuge briefly and discard the liquid.

[0089] Keep the 1.5 mL EP tube on the magnetic stand at all times, open the lid and air dry the magnetic beads until there is no ethanol residue; Remove the 1.5 mL EP tube from the magnetic stand and elute it.

[0090] (6) Transcriptome self-built library primer sequence

[0091] Example 4: Genomic sequencing library construction scheme Kit: Hieff NGS® OnePot Pro DNA Library Prep Kit V2 One-step DNA library preparation kit V2 (Cat No. 12195) (1) DNA fragmentation / end repair / dA tail addition This step fragments the genomic DNA, performs end repair and dA tail addition.

[0092] Thaw the reagents in the table, invert to mix, and place on ice for standby.

[0093] Prepare the reaction system in the table on ice.

[0094]

[0095] Mix gently with pipette or low speed vortex and centrifuge briefly to the bottom of the tube.

[0096] Place the PCR tube in the PCR instrument with a preset temperature of 4℃. When the module temperature drops to 4℃, place the PCR tube in the PCR instrument. Set the reaction program shown in the table below to perform the DNA fragmentation, end repair and dA tail addition reaction.

[0097]

[0098] (2) Adapter ligation 1) Reaction system:

[0099] 2) Mix gently with pipette or vortex and centrifuge briefly to collect the reaction solution to the bottom of the tube.

[0100] 3) Place the PCR tube in the PCR instrument and set the reaction program shown in the table below to perform the adapter ligation reaction.

[0101]

[0102] 4) Ligation product magnetic bead purification Use 0.9 times DNA Clean Beads for purification: Incubate at room temperature for 5 min; Centrifuge the 1.5 mL EP tube briefly and place it on the magnetic stand. After the solution is clear (about 5 min), carefully remove the supernatant; Keep the 1.5 mL EP tube on the magnetic stand at all times. Slowly add 200 μL of freshly prepared 80% ethanol to rinse the magnetic beads. Incubate at room temperature and carefully remove the supernatant; Repeat step 5 for a total of two rinses. Keep the 1.5 mL EP tube on the magnetic stand at all times. Open the cap and air dry the magnetic beads until there is no ethanol residue. Remove the 1.5 mL EP tube from the magnetic stand and elute.

[0103] (3) Nest PCR Reagent: Yousheng 2x Hieff Canace plus PCR Master Mix (with Dye) (10154) System as follows:

[0104] Reaction procedure:

[0105] (4) Index PCR Reagent: Yixing 2x Hieff Canace plus PCR Master Mix (with Dye) (10154) The system is as follows, and 10 parts of the system are used for purification using different amounts of beads:

[0106] Reaction procedure:

[0107] (5) Product magnetic bead purification 0.8 times DNA Clean Beads were used respectively (6) Primers for genomic library construction

[0108] Example 5: Phenotype information response transposon mutant library sequencing data bioinformatics analysis and phenotype result verification (1) Phenotype information response transposon mutant library sequencing data bioinformatics analysis According to the sequencing data obtained by the transposon mutant library experiment, the following bioinformatics analysis was performed: 1) The raw sequencing data obtained by the high-efficiency quality control software fastp was used for quality evaluation and filtering, and low-quality sequences and adapter contamination were removed to generate a high-quality cleaned data set; 2) Through the cutadapt tool, based on the specific sequence designed in the plasmid, read1 was screened and truncated, and the corresponding read2 sequence was extracted, realizing accurate cutting and enrichment of sequencing data; 3) Extract the pre-designed unique molecular identifier (UMI) from the retained read2 sequence, each read corresponds to a UMI, generate a UMI sequence information file, which is used for subsequent removal of PCR amplification bias and ensures quantitative accuracy; 4) Use the BWA (Burrows-Wheeler Aligner) tool to align read1 sequence to the reference genome (E. coli MG1655) to obtain the insertion site (pos) and direction information corresponding to each read, and output a standard SAM format file; 5) Integrating the alignment information of read1 (SAM), UMI information of read2 and raw sequence of read1, de-duplicating based on the matched UMI set of insertion site (pos) to ensure that each site only counts once for the insertion event from independent molecules, thus obtaining the unique template count of each site; 6) Using the reference genome annotation file (GFF format) published by NCBI, mapping each insertion site (pos) to the specific functional element in the genome (such as gene name, protein number, gene type, etc.), and determining whether it is located in the coding region or intergenic region; 7) Aggregating sites with functional annotations by gene name, counting the unique template UMI count of each gene to represent the transposon insertion abundance of each gene; 8) Integrating the results of multiple experimental conditions or different treatment groups, constructing a standardized insertion abundance matrix to realize further research such as functional comparison, insertion hotspot analysis or lethal gene screening across samples, and the specific analysis process is shown in FIG. 8. Figure 10

[0109] (2) Knockout of phenotype-related genes According to the target phenotype list, the inventors selected 15 genes for scarless knockout using CRISPR / Cas9 technology. After knockout, the tryptophan fermentation of these single-knockout strains was performed and the tryptophan content was detected by ultra-high throughput mass spectrometer. The information of the 15 genes and gRNA sequences are shown in the following table. The following tryptophan-related genes are owned by the present application.

[0110] Table 5 Tryptophan phenotype-related genotypes and gRNA sequences

[0111] (3) Fermentation and detection of phenotype-related genes After successfully knocking out the above genes alone, the tryptophan shake flask fermentation of these knockout strains and the starting strain Escherichia coli K-12 MG1655 ΔendA ΔrecA (DE3) strain was performed. First, single colonies of knockout strains and starting strains (three biological replicates) were picked and inoculated in 2 mL tryptophan fermentation liquid medium at 37°C overnight to obtain seed liquid. Then, the seed liquid was inoculated in 100 mL triangular flask containing 20 mL tryptophan fermentation liquid medium, and the shake flask fermentation was carried out at 37°C for 36 h. After that, the intracellular and extracellular tryptophan was extracted, and finally the ultra-high throughput mass spectrometer Echo MS was used to detect the intracellular and extracellular tryptophan. The detection results are shown in FIG. 9. Figure 11 The relative value of intracellular ΔrcsB, ΔsspB, ΔmiaA, ΔidnD strain tryptophan is more than one time of the starting strain, and the extracellular ΔrcsD, ΔrcsB, ΔptsP ​The relative value of tryptophan of the strain is more than one times of the starting strain. The data shows that the present application can directly obtain the gene related to the tryptophan phenotype information through transcription level without modifying the strain (the starting strain is close to the wild type strain).

[0112] While embodiments of the present application have been shown and described with reference to particular embodiments thereof, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the application. The scope of the application is defined by the appended claims and their equivalents.

Claims

1. A method for quantitative phenotyping of genotypes based on transposon mutagenesis and high-throughput sequencing, characterized in that, The method comprises the following steps: design and construct a recording transposon and a temperature-sensitive transposition plasmid; introduce the temperature-sensitive transposition plasmid into a host strain to obtain a transposition strain, induce the recording transposon to insert into the host genome and perform plasmid loss through temperature regulation, verify and evaluate after expansion culture, and obtain a verified transposon mutant library; inoculate the transposon mutant library into a culture system corresponding to a target phenotype, convert the genotype difference caused by the insertion of the recording transposon into a phenotype difference, convert the phenotype difference into a sequencable fusion transcript through a biological sensor of the recording transposon, and collect bacterial cells after the culture ends; extract total DNA and total RNA of the bacterial cells, and construct a genomic DNA library for locating genotypes and an RNA-cDNA library for quantifying phenotypes; perform high-throughput sequencing on the genomic DNA library and the RNA-cDNA library, perform bioinformatics analysis to locate the insertion site of the recording transposon, quantify the fusion transcript, and correlate the genotype and the phenotype, and screen key candidate genes for regulating the target phenotype; perform scarless knockout on the key candidate genes, and verify the regulation function of the genes on the target phenotype through fermentation experiments and phenotype detection.

2. The transposon mutagenesis and high-throughput sequencing based genotypic phenotypic quantitative characterization method according to claim 1, characterized in that: The recording transposon comprises a transposition mediation unit, a screening marker unit and a phenotype recording unit; the transposition mediation unit is a Mariner himar1 C9 transposase-specific inverted repeat sequence at the left and right ends of the recording transposon, the Mariner himar1 C9 transposase is a variant optimized through directional mutation, and only a 5'-TA-3' sequence is inserted into the host genome; the screening marker unit is a spectinomycin resistance gene in the middle region of the recording transposon; the phenotype recording unit is an integrated phenotype response type biological sensor, the biological sensor is composed of a regulatory protein coding gene and a reporter gene, and no transcription terminator is arranged downstream of the reporter gene, so that transcription can be read-through from the biological sensor promoter to the host genome downstream of the transposon insertion site, forming a sensor-host genome fusion transcript.

3. The transposon mutagenesis and high-throughput sequencing based genotypic phenotypic quantitative characterization method according to claim 1, characterized in that: The temperature-sensitive transposition plasmid takes a plasmid containing a temperature-sensitive replicon as a backbone, is used for carrying the recording transposon, and performs subsequent plasmid loss.

4. The transposon mutagenesis and high-throughput sequencing based genotypic phenotypic quantitative characterization method according to claim 1, characterized in that: The construction of the transposon mutant library specifically comprises the following steps: transposition strain preparation: introduce the temperature-sensitive transposition plasmid into the host strain by using an electroporation method, coat an LB solid plate containing ampicillin after electroporation, culture at 30 DEG C for 16 hours, pick single colonies to obtain the transposition strain; transposition induction: inoculate the transposition strain single colonies into an LB liquid culture medium containing ampicillin, obtain a seed liquid through overnight culture, transfer the seed liquid into an LB liquid culture medium containing spectinomycin and IPTG, perform overnight culture at 16 DEG C to induce the expression of the transposase, and obtain a bacterial liquid; plasmid loss and library expansion: transfer the bacterial liquid into an antibiotic-free LB liquid culture medium, perform overnight culture to perform plasmid loss, and then transfer into an LB liquid culture medium containing spectinomycin, and perform overnight culture to expand the library; Library preservation: take the bacteria liquid after expansion culture, add 50% glycerol at a volume ratio of 1:1, and store at -80℃ to obtain the transposon mutant library.

5. The transposon mutagenesis and high-throughput sequencing based genotypic phenotypic quantitative characterization method according to claim 1, characterized in that: The verification evaluation specifically includes: Insertion randomness verification: using Tail PCR method, taking the genomic DNA of the transposon mutant library as template, using transposon-specific nested primers and random degenerate primers for three rounds of nested PCR, sequencing the PCR products and aligning to the host genome to verify randomness; Whole genome coverage verification: using DNA footprinting method, designing specific primer pairs of essential genes, non-essential genes and random genes, verifying the coverage of the transposon on the whole genome through the distribution of PCR product bands.

6. The transposon mutagenesis and high-throughput sequencing-based genotypic phenotypic quantitative characterization method according to claim 2, characterized in that: The phenotype-responsive biosensor is used for sensing the intensity of intracellular specific molecular signal, and when the target phenotype is tryptophan metabolism phenotype, the phenotype-responsive biosensor is composed of tnaC The gene is fused with an enhanced green fluorescent protein gene.

7. The transposon mutagenesis and high-throughput sequencing-based phenotyping method of genotypes of claim 1, characterized in that: The construction of the genomic DNA library includes the following steps: Fragmentation, end repair and dA tail addition are performed on the extracted total DNA to form dA tail-modified DNA fragments; A specially designed UMI adapter containing a unique molecular identifier is added to the dA tail-modified DNA fragments, and a ligase is added for adapter ligation reaction. After the reaction is completed, the ligation product is obtained by magnetic bead purification; Using the ligation product as template, Nest PCR is performed using transposon-specific primers and sequencing adapter primers to enrich the Nest PCR amplification product containing transposon flanking sequences; Using the Nest PCR product as template, Index PCR is performed by adding Index primers with sample distinguishing tags. After the reaction is completed, the genomic DNA library is obtained by magnetic bead purification.

8. The transposon mutagenesis and high-throughput sequencing-based genotypic phenotypic quantitative characterization method according to claim 1, characterized in that: During the construction of the RNA-cDNA library, a unique molecular identifier is introduced in the reverse transcription stage, which is used to remove PCR amplification bias in data analysis and count the original number of fusion transcripts.

9. The transposon mutagenesis and high-throughput sequencing-based phenotyping method of genotypes of claim 1, wherein: High-throughput sequencing is performed using the Illumina NovaSeq 6000 platform for double-end sequencing. The bioinformatics analysis includes: using BWA software to align the sequencing data of the genomic DNA library to the host reference genome to locate the transposon insertion site, using DESeq2 software to perform differential analysis on the quantitative data of fusion transcripts, and screening to obtain key candidate genes regulating the target phenotype.

10. The transposon mutagenesis and high-throughput sequencing-based phenotyping method of genotypes of claim 1, wherein: The scarless knockout uses CRISPR / Cas9 technology. After cloning the specific gRNA of the key candidate gene into the pCas9 vector, it is introduced into the host strain to induce Cas9 protein expression and achieve gene knockout. The phenotype detection uses an ultra-high-throughput mass spectrometer to detect the content of target substances in the fermentation product to verify the regulatory function of the knockout gene on the target phenotype.

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