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Oct 2019
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Defined Mutant Library Sequencing (DML-Seq) for Identification of Conditional Essential Genes
确定突变体文库测序(DML-Seq)用于鉴定的条件必需基因   

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Abstract

Transposon insertion sequencing (TIS) is an emerging technique which utilizes a massive transposon mutant library to screen specific phenotype and determine the conditional essential genetic requirements for bacterial fitness under distinct conditions combined with high-throughput parallel sequencing technology. Compared with a massive mutant library in traditional TIS, the defined mutant library sequencing (DML-Seq) has advantages as: 1) efficient mutagenesis; 2) low bottleneck effects; 3) avoid hotpots caused by screening; 4) can be directly used in the following experiments. Here, we described an optimized procedure of DML-Seq for fitness screen to supply classical TIS using the marine pathogenic bacterium Edwardsiella piscicida as an example.

Keywords: TIS (转座子插入测序), DML-seq (确定突变体文库测序), Defined mutant library (确定突变体库), Conditional essential genes (条件性必需基因)

Background

Transposon insertion mutagenesis coupled with next-generation sequencing (NGS) has been proven to be a powerful method to investigate gene function under a multitude of conditions (Chao et al., 2016; Price et al., 2018). In general, TIS analysis consists of the massive parallel sequencing of transposon insertion sites and statistical analysis of the abundance of the insertion events.


TIS-based screens can provide high-resolution maps of the fitness contributions of individual loci and domains based on the bacterial fitness of highly saturated transposon mutant libraries under a multitude of conditions (Chao et al., 2016). The insertion frequency at each locus or the relative abundance of corresponding mutants is generally inversely correlated with the locus’s contribution to fitness following the imposition of a selective pressure, such as that imposed by hosts and antibiotics (Chao et al., 2016). The principles of this methodology underlie a variety of related approaches, including TIS, transposon sequencing (TnSeq), insertion sequencing (INSeq), transposon-directed insertion site sequencing (TraDIS), high-throughput insertion tracking by deep sequencing (HITS), and transposon insertion site sequencing (TIS-Seq) (Gawronski et al., 2009; Langridge et al., 2009; Chao et al., 2016; Veeranagouda et al., 2017; Price et al., 2018). These approaches are revolutionizing microbiological studies by facilitating unprecedented and deep genome-wide explorations of a wide range of bacterial species.


With the aim to generate highly saturated mutant libraries for further analysis, the insertion events caused by transposon should be unbiased and the transposon will be delivered into recipient cells several times. However, this will lead to a large amount of nonsense mutation and the complexity of mutant libraries may cause bottleneck effect during the following screens. Compared with this, the defined mutant libraries containing mutants with a single insertion at a known genomic location will be more useful and facilitated (Fu et al., 2013; Abel et al., 2015). The first human pathogenic bacterium defined mutant library for Pseudomonas aeruginosa was constructed in 2003. Subsequently, many defined mutant libraries of human pathogens were also generated, such as a library of Francisella tularensis in 2007, Vibrio cholera in 2008, Burkholderia thailandensis in 2013, Chlamydia and Mycobacterium in 2015 (Jacobs et al., 2003; Gallagher et al., 2007 and 2013; Cameron et al., 2008; Kokes et al., 2015). With these defined mutant libraries, a myriad of important researches were completed, such as the identification of ciprofloxacin resistome from P. aeruginosa library, a novel T6SS regulator TsrA from V. cholera library, and the exploration of functional genomics with Desulfovibrio alaskensis G20 library (Breidenstein et al., 2008; Zheng et al., 2008 and 2010; Kuehl et al., 2014). Hence, such defined libraries may serve as valuable resources because they often consist of collections of insertion mutants in almost all non-essential loci for an organism of interest and have drawn more attention to the exploration of functional genomics of microbe.


In order to optimize TIS approach, we developed the DML-seq method which adopts the mixed defined mutant libraries for conditional essential fitness screenings (Figure 1) (Wei et al., 2019a and 2019b). Compared with massive mutant library in traditional TIS, the DML library has advantages as: 1) insertions within the 80% of the ORF to ensure the effective mutagenesis; 2) low complexity and few isogenes to ensure low bottleneck effects; 3) the mixture of an equal amount of individual mutants to avoid hotpots caused by screening; 4) can be directly used in the following experiments.


Compared to human pathogenic bacterium, the pathogenesis of marine pathogenic bacterium is poorly understood. In our model organism Edwardsiella piscicida, which causes Edwardsiellosis in farmed fish and leads to severe economic losses in aquaculture worldwide (Leung et al., 2019), DML-seq has been employed to identify conditional essential genes as well as those crucial for survival in different environments (Wei et al., 2019b), colonization and infection in host cells (Wei et al., 2019a). Briefly, we constructed MKGR, a derivative of Himar1 mariner transposon based on MAR2xT7 (Liberati et al., 2006), to generate a library of random transposon insertion mutants of E. piscicida EIB202 (Table 1). In total, 2,806 genes were disrupted out of the 3,599 predicted genes with an average of 5.04-fold coverage on E. piscicida chromosome. After that, 5 non-redundant subset libraries were created manually. The 1st and 2nd subset libraries contained a single mutant for each disrupted ORF in the parental library and they were paralleled for each other. The 3rd subset library contains transcriptional fusion mutants to eliminate the location effect on transcription. The 4th subset library is an intergenic library and the 5th library was a composited library, which was generated by mixing the 1st, 2nd, 4th subset libraries into a pool.


Here, we will focus on the specific steps for DML-seq because the excellent protocols deTAILing TIS analyses are reviewed and available elsewhere (Chao et al., 2016; Yamaichi et al., 2017). In principle, the procedure described here can be applied to conditional essential screens and facilitate the exploration of functional genomics of microbe.


Table 1. The statistic values of defined mutant library of E. piscicida EIB202




Figure 1. A defined transposon mutant library construction. A. pMKGR, a derivative of Himar1 mariner transposon, was constructed to build the defined transposon mutant library of E. piscicida EIB202. B. Work flow of a defined mutant library construction. E. coli SM10 is used as a donor for conjugation with recipient strain E. piscicida EIB202. Conjugation is processed between EIB202 and SM10 on LB plates for 3 h at 30 °C. Later, conjugators are separated on LB plates containing 20 µg/ml polymyxin and 15 µg/ml gentamicin. After 24 h of incubation at 30 °C, monoclonal will be picked into 96-well plates and incubated at 30 °C for about 16 h. Before storing at -80 °C, glycerol was added into every well and mix to make the final 20% glycerol concentration to preserve strains. C. Schematic of TAIL-PCR amplification used for determination of the Tn insertion loci on the genome. The pair of primer Sp1/AB2 is used for the first round of PCR and another pair of primer Sp2/ABS is used for the second round of PCR. Finally, primer Seq2 is used for sequencing of the amplified PCR products. D. The number of new disrupted genes increased linearly with new mutants and the number of genes hit approached a plateau.


Materials and Reagents

  1. Handling Bacteria

    1. Large square Petri dish (500 cm2) (Thermo Fisher Scientific, catalog number: R80115TS)

    2. Cellulose filter membrane (0.45 μm pore size, Merck Millipore)

    3. Eppendorf tube (Axygen, catalog number: MCT-150-C)

    4. PCR tube (Axygen, catalog number: PCR-02-A)

    5. Corning 50 ml centrifuge tube (Merck, catalog number: CLS430828-500EA)

    6. 96-Well Deep Well Plates (Thermo Fisher Scientific, catalog number: A43075)

    7. Cryotube (Thermo Fisher Scientific, catalog number: 5000-1020)

    8. Wild type E. piscicida EIB202 (ColR); “recipient” cells

    9. E. coli SM10 λpir harboring pMKGR for delivering Tn via conjugation (AmpR, KmR); “donor” cells (see Note 1)

    10. Luria-Bertani (Miller's LB Broth Base, Thermo Fisher Scientific, catalog number: 12795027) and agar media (Merck, catalog number: 05040)

    11. Colistin (Col, 20 mg/ml, BBI)

    12. Ampicillin (Amp, 100 mg/ml, BBI)

    13. Gentamicin (Gm, 15 mg/ml, BBI)

    14. Glycerol (Titan, catalog number: 56-81-5)


  2. Thermal Asymmetric Interlaced PCR (TAIL-PCR)

    1. Tiangen 2× Taq PCR MasterMix (TIANGEN BIOTECH, catalog number: KT205-02)

    2. Nuclease-free water H2O (QIAGEN, catalog number: 129114)


  3. Genomic DNA (gDNA) Purification

    1. 0.5 ml individual tubes (Thermo Fisher Scientific, catalog number: AB0350)

    2. Tiangen gDNA extraction kit (TIANGEN BIOTECH, catalog number: DP302-02)

    3. 100% ethanol (Titan, catalog number: 64-17-5)


  4. Library Construction

    1. Qiagen Gel purification kit (QIAGEN, catalog number: 28104)

    2. VAHTS HiFi Amplification Mix (Vazyme Biotech, catalog number: 616-01/02)

    3. Agarose (Thermo Fisher Scientific, catalog number: 16500500) and Electrophoresis apparatus (BIO-RAD, catalog number: 1704489EDU)

    4. QubitTM dsDNA HS Assay kit (Invitrogen, catalog number: Q32851)


  5. Oligo DNAs (See Note 2) (HPLC purified, BGI)

    1. “EIB202-F”: 5’-TCATCGCACATACAGAATAAACGCC-3’

    2. “EIB202-R”: 5’-CCGTAACATTTCTTACAACACTGCG-3’

    3. “pMKGR-F”: 5’-AGGTGATGCTACATACGGAAAG-3’

    4. “pMKGR-R”: 5’-AGCGCATGAACTCCTTGATG-3’

    5. “Tn-F”: 5’-AAAAGTCCGCTGGCAAAG-3’

    6. “Tn-R”: 5’-CCCTTCAAGAGCGATACAAC-3’

    7. “Sp1-F”: 5’-GCTCCGTAGTAAGACATTCATCGCG-3’

    8. “Sp2-F”: 5’-GCTTACGTTCTGCCCAAGTTTGAG-3’

    9. “ABS-R”: 5’-GGCCACGCGTCGACTAGTAC-3’

    10. “AB2-R”: 5’-GGCCACGCGTCGACTAGTACNNNNNNNNNNCCTGG-3’

    11. “Seq2-F”: 5’-CAATTCGTTCAAGCCGAGATCG-3’

    12. “AD_fork truncated NH2”: 5’- TACCACGACCA-3’

    13. “AD_Index Fork R”: 5’- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATGGTCGTGGT-3’

    14. “1st_seq-out-psc189”: 5’- ACTCTGGGGTACGCGTCTAG-3’

    15. “1st_PCR_Index ‘R’ primer”: 5’-GTGACTGGAGTTCAGACGTGTG-3’

    16. “P5 spacer primers”: equimolar mixture of the following 6 oligos:

      5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGACTTATCAGCCAACCTGT-3’

      5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCGACTTATCAGCCAACCTGT-3’

      5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTATGACTTATCAGCCAACCTGT-3’

      5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGTCGACTTATCAGCCAACCTGT-3’

      5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTCGACGACTTATCAGCCAACCTGT-3’

      5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGCAGCGACGACTTATCAGCCAACCTGT-3’

    1. “P7 barcode primers”

      CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC (vary from sample to sample)

Equipment

  1. Multichannel pipette (Thermo Fisher Scientific)

  2. High speed centrifuge (Eppendorf, model: 5415R)

  3. Thermal cycler (EASTWIN, model: ETC811)

  4. Water bath (Thermo Fisher Scientific, catalog number: TSGP02)

  5. High speed centrifuge (Eppendorf, model: 5415R)

  6. Bath sonicator (Diagenode, catalog number: B01020001)

  7. Thermal cycler (EASTWIN, catalog number: ETC811)

  8. VAHTS Universal End preparation Module for Illumina (Vazyme Biotech, catalog number: N203-01)

  9. VAHTS Universal Adapter Ligation Module for Illumina (Vazyme Biotech, catalog number: N204-01)

  10. QubitTM 4 Fluorometer (Invitrogen, catalog number: Q33238), or equivalent fluorescent-based DNA quantification apparatus

Procedure

  1. Parental Library Construction

    1. Inoculate “donor” (in LB broth with Amp, 100 μg/ml) and “recipient” cells (in LB broth with Col, 20 μg/ml) at 37 °C with shaking and culture overnight.

    2. Prepare LB agar plate(s) without antibiotics. Let them dry well and place 0.45 μm membrane filters on top of the plates (four membranes can be applied to each plate) (see Note 3).

    3. Passage E. piscicida, 2 ml in 50 ml (4%), add CaCl2 to 50 mM (500 μl, 10 M), Rotate 200 rpm at 30 °C, the OD600 will reach ~0.6-0.8 after 1 h 15 min culture (see Note 4).

    4. Measure the OD600 of all bacteria, which are expected to be 0.8, for SM10 and E. piscicida, respectively.

    5. Move the culture into 50 ml corning tubes, spin 5,000 × g at 10 °C for 5 min, then wash with 10 ml LB and spin 5,000 × g for 10 min.

    6. Resuspend in LB with the following volume to reach final OD600 = 4.

    7. Mix equal volume of SM10 and E. piscicida in an Eppendorf tube, and spot 4*80 μl on the filter prepared in Step A2. Let drop adsorb into the agar and dry.

    8. Place these plates into a 30 °C incubator with care not to let the cell suspension spill outside the membrane (do not flip the plate).

    9. After 3 h of incubation, remove the filter into a 50-ml centrifuge tube by forceps. Resuspend cells from filters in 1 ml of LB broth by pipetting up/down and vortexing.

    10. Make serial dilutions of resuspended cells and plate 100 µl of 1:1, 1:10, and 1:100 dilutions onto LB agar plates containing Col and Gm (to provide an estimate of transposon insertion efficiency, as the actual library plates can become too crowded to pick colonies). Incubate overnight at 30 °C.

    11. Examine ~100 ColR GmR colonies by colony PCR with 3 pairs of universal primers EIB202-F/R, pMKGR-F/R, and Tn-F/R (see Note 5).

    12. Pick monoclonal into 96 deep well plates, rotate 200 rpm at 30 °C overnight.

    13. Mix 900 µl of cells with 300 µl of 80% glycerol into cryotube to store a recoverable library at -80 °C.


  2. Thermal Asymmetric Interlaced PCR (TAIL-PCR)

    1. For each monoclonal, prepare the first reaction system in a PCR tube:

      0.5 µl bacterial culture (from Subheading 3.1).

      1 µl 10 µM “Sp1-F”.

      1 µl 10 µM “AB2-R”.

      10 µl Tiangen 2× Taq PCR MasterMix.

      Add H2O to 20 µl.

    2. Run on the PCR Thermo cycler under the program.

      1 = 94 °C for 4 min.

      2 = 94 °C for 30 s.

      3 = 42 °C for 30 s.

      [option] -1 °C per cycle.

      4 = 72 °C for 3 min.

      5 = go to 2, 6 times.

      6 = 94 °C for 30 s.

      7 = 58 °C for 30 s.

      8 = 72 °C for 1 min.

      9 = go to 6, 25 times.

      10 = 72 °C for 3 min.

    3. Dilute the PCR product ten-fold, prepare the second reaction system in a PCR tube:

      1 µl Dilute PCR products.

      2 µl 10 µM “Sp2-F”.

      2 µl 10 µM “ABS-R”.

      25 µl Tiangen 2× Taq PCR MasterMix.

      Add H2O to 50 µl.

    4. Run on the PCR Thermo cycler under the program.

      1 = 94 °C for 3 min.

      2 = 94 °C for 30 s.

      3 = 64 °C for 30 s.

      4 = 72 °C for 1 min.

      5 = go to 2, 30 times.

      6 = 72 °C for 3 min.

    5. Collect the PCR product and sequence with primer “Seq2”.


  3. Non-redundant Subset Library Construction (see Note 6, E. piscicida EIB202 used as an example)

    1. Sequence results are mapped to recipient EIB202 genome.

    2. Saturation curve is drawn to ensure the saturation of the library.

    3. According to identified insertion sites, non-redundant subset libraries were generated manually to facilitate a genome-wide screen.

    4. According to different experimental purposes, non-redundant subset libraries would be used for the screen.


  4. gDNA Extraction

    Use commercial gDNA extraction kit, refer to relevant protocol.


  5. gDNA Shearing by Sonication

    1. Dilute gDNA in fresh 0.5 ml individual tubes to a final concentration of 50 ng/µl in 100 µl H2O.

    2. Sonicate DNA to yield fragments: 30s ON/ 90s OFF, for 12 cycles.

    3. Run a sample (5 µl) of the digest on an agarose gel (2%). Most of the DNA should fall in the range of 200-500 bp, which will be fine for downstream ligations and PCR.


  6. End Repair and A-TAILing

    1. Prepare the reaction in a PCR tube:

      VAHTS Turbo End Prep Enzyme Mix: 3.0 µl.

      VAHTS Turbo End Prep Reaction Buffer (10×): 6.5 µl.

      Fragmented DNA (50 ng/µl, add 1 µg): 20 µl.

      Add H2O to 65.0 µl.

    2. Carry out PCR using the following cycling conditions.

      1 = 20 °C for 30 min.

      2 = 65 °C for 30 min.


  7. Adapter Ligation

    1. Prepare the following adapter mixture in a PCR tube.

      5 µl 100 µM “AD_fork truncated NH2”.

      5 µl 100 µM “AD_Index Fork R”.

      0.4 µl 2 mM MgCl2.

    2. Run on the PCR Thermo cycler under the program.

      1 = 95 °C for 4 min.

      2 = 95 °C for 1 min.

      [option] -1 °C per cycle.

      3 = go to 2, 75 times.

      4 = End.

    3. Dilute to half concentration: Add the same volume of H2O.

    4. Prepare the following ligation reaction in a PCR tube.

      A-TAILed DNA (from above Subheading 3.5): 65 µl.

      VAHTS Turbo T4 DNA Ligase: 2.0 µl.

      VAHTS Turbo Ligation Enhancer: 30.5 µl.

      Adapter for Tn-seq (from above Subheading 3.6, step 2): 2.5 µl.

      Add H2O to 100 µl.

    5. Incubate the reaction at 20 °C for 30 min.

    6. Column-purify DNA, elute with 35 µl pre-warmed H2O.


  8. Amplification of Tn-associated gDNA

    1. Prepare the following PCR reaction in an Eppendorf tube.

      50 µl VAHTS HiFi Amplification Mix.

      200 ng Ligated DNA (from Subheading 3.6).

      5 µl 10 µM “1st_seq-out-psc189 primer”.

      5 µl 10 µM “1st_PCR_Index ‘R’ primer”.

      Add H2O to 100 µl.

    2. Aliquot into 2 PCR tubes (50 µl each) and carry out PCR using the following cycling conditions.

      1 = 98 °C for 30 s.

      2 = 98 °C for 10 s.

      3 = 53 °C for 30 s.

      4 = 72 °C for 30 s.

      5 = go to 2, 29 times in all.

      6 = 72 °C for 5 min.

    3. Pool the 2 PCR reactions and then column-purify the PCR products, elute with 35 µl pre-warmed H2O.


  9. Second PCR to Add barcodes, Illumina Attachment (P5, P7) and Variability Sequences

    1. Prepare the following PCR reaction in an Eppendorf tube.

      300 ng purified PCR product (from Subheading 3.7).

      75 µl VAHTS HiFi Amplification Mix.

      7.5 µl 10 µM “P5 spacer primers”.

      7.5 µl 10 µM P7 Index barcode (should vary sample by sample).

      Add H2O to 150 µl.

    2. Aliquot into 3 PCR Tubes (50 µl each) and carry out PCR using the following cycling conditions.

      1 = 98 °C for 30 s.

      2 = 98 °C for 10 s.

      3 = 55 °C for 30 s.

      4 = 72 °C for 30 s.

      5 = go to 2, 17 times total.

      6 = 72 °C for 7 min.

    3. Pool the 3 PCR reactions and then column-purify the PCR products, elute with 35 µl pre-warmed H2O.


  10. Size Selection

    1. Run all the purified PCR products (from Subheading 3.8) on a 2% agarose gel in 0.5× TAE buffer.

    2. Size select by cutting out the smear between 200 and 500 bp. Make sure not to include the visible head-to-head primer dimers around 200 bp.

    3. Column-purify DNA using a standard gel extraction procedure, elute with 50 µl pre-warmed H2O.

    4. Quantify the DNA concentration by Qubit. Now the DNA is ready to be run on an Illumina sequencer.


  11. Conditional Essential Genes analysis

    The transposition loci and the abundance of the insertions are determined using the EL-ARTIST pipeline (Chao et al., 2013). Based on the neutral base pair model, a random distribution of transposon insertions, a library containing n mutants, and the total genes number on the genome is k, the number of genes uncovered by transposon is N. The probability of gene j getting insertion pj, the uncovered genes number by transposon is  The essential genes number is set as k1. As neutral base pair model, the probability of essential genes uncovered  and the predicted essential genes number k1 = N - Nt + Ne.

Notes

  1. pMKGR was constructed based on Himar1 mariner transposon. The promoterless kanamycin resistance (Kanr) cassette and gene egfp with ribosomal binding site (RBS) were amplified. The RBS sequence used in this study was “AAGGAGG”, which originated from the E. piscicida 16S rRNA 3’ terminal conserved sequence “CCUCCUU”. A triple terminal sites “TGACTAGCTAA” and 48-bp T7 terminator sequence were introduced into pMKGR. The constructed pMKGR was transformed into E. coli SM10 and validated by PCR and sequencing.

  2. The pMKGR, a derivative of Himar1 mariner transposon and strain E. piscicida EIB202 are used as examples in this protocol; for different transposons and strains, all primer sequences need to be modified accordingly.

  3. The prepared membranes should be as many as conjugation reactions. With more and more picked colonies for sequencing, the number of disrupted genes is increasing and the increasing rate becomes horizontal gradually and reaches a plateau in Step C2. It means that plenty of mutagenesis has already been generated and collected mutants are able to assemble a comprehensive and saturated library.

  4. The optimal growth temperature of E.piscicida EIB202 is 30 °C. The following incubation temperature needs to be modified according to different recipient strains.

  5. When antibiotics are used as selective markers, there is a small chance to observe colonies that are not truly resistant and can grow in the presence of antibiotics even without containing a transposon insertion (phenotypic resistance). To ensure successful transposition, it is suggested to randomly restreak ~100 colonies on fresh plates and perform colony PCR with 3 pairs of universal primers which are designed for transposon, its vector plasmid and “recipient” cells. For colony PCR, 1) Pick a single colony with a sterile flat toothpick or pipette tip and swirl in sterile water. 2) Lyse the bacteria to release DNA by either simply boiling the sample before use or directly adding a small volume of the sample to the PCR reaction. The bacteria will be lysed at the initial heating step of the conventional PCR procedure. 3) The cycling conditions are: 98 °C, 1 min; followed by 30 cycles of 98 °C, 5 s; 55 °C, 5 s; and 72 °C, 40 s. 4) The size of PCR products is determined by electrophoresis and then PCR products are verified with Sanger Sequencing.

  6. Five subset libraries were separated from the parental library. The 1st and parallel 2nd subset libraries include a single mutant for each ORF, respectively and insertion sites at 0.4-0.6 percentage within each ORF 5’-3’ end were given priority. Once the ORF contains only one insertion site, these mutants were selected prior into 1st subset library. The 3rd subset library is a transcriptional fusion library in which each mutant carries the same orientation of MKGR with local ORF and the priority was given to insertion sites near the 5’ end of each ORF. Similar to the 1st subset library criteria, the 4th library was generated and includes no more than two mutants for each intergenic region. The 5th compound library is the mixture of all the mutants from the 1st, 2nd and 4th subset libraries and takes 2 x 107 CFU for each corresponding mutant.

Acknowledgments

The described protocol is adapted from Wei et al., 2019. This work was supported by grants from the National Natural Science Foundation of China (32002436 to S.S., 82001033 to L.W.), National Key Research and Development Program of China (2018YFD0900504 to Q.W.), Shanghai Sailing Program (20YF1411100), China Postdoctoral Science Foundation (2020M671034), Ministry of Agriculture of China (CARS-47), and the Science and Technology Commission of Shanghai Municipality (17391902000).

Competing interests

There are no conflicts of interest or competing interest.

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  16. Price, M. N., Wetmore, K. M., Waters, R. J., Callaghan, M., Ray, J., Liu, H., Kuehl, J. V., Melnyk, R. A., Lamson, J. S., Suh, Y., Carlson, H. K., Esquivel, Z., Sadeeshkumar, H., Chakraborty, R., Zane, G. M., Rubin, B. E., Wall, J. D., Visel, A., Bristow, J., Blow, M. J., Arkin, A. P. and Deutschbauer, A. M. (2018). Mutant phenotypes for thousands of bacterial genes of unknown function. Nature 557(7706): 503-509.
  17. Veeranagouda, Y. and Didier, M. (2017). Transposon Insertion Site Sequencing (TIS-Seq): An Efficient and High-Throughput Method for Determining Transposon Insertion Site(s) and Their Relative Abundances in a PiggyBac Transposon Mutant Pool by Next-Generation Sequencing. Curr Protoc Mol Biol 120: 21 35 21-21 35 11.
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简介

[摘要]转座子插入测序(TIS)是一项新兴技术,它利用大量的转座子突变体文库筛选特定表型,并结合高通量并行测序技术,在不同条件下确定细菌适应性的条件性基本遗传要求。与传统TIS中的大规模突变文库相比,已定义的突变文库测序(DML-Seq)具有以下优势:1)高效诱变;2)瓶颈效应低;3)避免因筛选引起的火锅;4)可直接用于以下实验。在这里,我们描述DML-SEQ的优化过程进行健身屏幕使用海洋致病菌提供古典TIS爱德华piscicida作为一个例子。


[背景]转座子插入诱变与下一代测序(NGS)结合已被证明是在多种条件下研究基因功能的有效方法(Chao等,2016; Price等,2018)。通常,TIS分析由转座子插入位点的大规模平行测序和大量插入事件的统计分析组成。

基于TIS的筛选可以在多种条件下基于高度饱和的转座子突变体文库的细菌适应性,提供单个基因座和域的适应性贡献的高分辨率图(Chao等,2016)。每个位点的插入频率或相应突变体的相对丰度通常与施加选择性压力(例如宿主和抗生素施加的压力)后与基因座对适应性的贡献成反比(Chao等人,2016)。这种方法的原理是多种相关方法的基础,包括TIS,转座子测序(TnSeq ),插入测序(INSeq ),转座子定向插入位点测序(TraDIS ),高通量深度测序跟踪插入(HITS)和转座子插入位点测序(TIS-Seq)(Gawronski等,2009; Langridge等,2009; Chao等,2016; Veeranagouda等,2017; Price等,2018)。这些方法通过促进对各种细菌物种的前所未有的深入的全基因组探索,正在彻底改变微生物学研究。

为了生成高度饱和的突变体文库以进行进一步分析,应公正对待转座子引起的插入事件,并将转座子多次递送至受体细胞中。然而,这会导致大量的无义突变和突变体库的复杂性可能会导致在瓶颈效应的如下画面。与此相比,定义的突变体文库将更有用和更容易获得,该文库包含在已知基因组位置具有单个插入的突变体(Fu等人,2013;Abel等人,2015 )。2003年建立了第一个铜绿假单胞菌的人类致病菌定义突变文库。随后,还产生了许多人类致病菌的定义突变文库,例如2007年的土拉弗朗西斯菌,2008年的霍乱弧菌,2013年的Burkholderia thailandensis ,衣原体的衣原体。和分枝杆菌(Mycobacterium )在2015年(Jacobs等,2003; Gallagher等,2007和2013; Cameron等,2008;Kokes等,2015 )。与这些定义突变体文库,重要的研究无数完成,如鉴定环丙沙星resistome从绿脓杆菌库,一个新颖T6SS调节器TSRA从霍乱弧菌库,和功能基因组学与勘探脱硫alaskensis G20库(Breidenstein等,2008;Zheng等,2008和2010; Kuehl等,2014 )。因此,这样定义的文库可以用作有价值的资源,因为它们通常由感兴趣的生物的几乎所有非必需基因座中的插入突变体集合组成,并引起了人们对微生物功能基因组学探索的更多关注。

为了优化TIS方法,我们开发了DML-seq方法,该方法采用混合定义的突变体文库进行条件基本适应性筛选(图1)(Wei等人,2019a和2019b)。与传统的大型突变体库相比TIS,DML库具有以下优点:1)在ORF的80%内插入以确保有效诱变;2)低复杂度和很少的同基因异构体,以确保低瓶颈效应;3)的混合物的等量个体突变体引起的通过筛选避免火锅的; 4)可直接用于以下实验。

与人类致病细菌相比,人们对海洋致病细菌的发病机理了解甚少。在我们的模型生物爱德华piscicida ,导致爱德华氏养殖鱼类,并导致水产养殖全球严重的经济损失(梁等人,2019 ),DML-SEQ已被用来识别条件性必需基因以及那些至关重要的生存不同的环境(Wei等人,2019b),在宿主细胞中的定植和感染(Wei等人,2019a)。简言之,我们构建MKGR,的衍生物Himar1水手转座子基于MAR2xT7(Liberati等人,2006 ),以生成的随机转座子插入突变体文库E. piscicida EIB202(表1 )。总共3,599个预测基因中,共有2,806个基因被破坏,平均在E. piscicida染色体上的覆盖率为5.04倍。之后,手动创建了5个非冗余子集库。1日和2个子集库包含在单个突变体为每个中断ORF的亲本文库和它们平行于彼此。3次亚库包含转录融合突变体,消除对转录的位置效果。4个子组库是基因间库和5个库是一个合成后的库,这是通过混合1产生ST ,2次,4次子集文库成池。

在这里,我们将专注于DML-seq的具体步骤,因为对TIS分析进行了详细研究的优秀协议已在其他地方进行了评论(Chao等,2016;Yamaichi等,2017 )。原则上,此处描述的过程可以应用于有条件的必要筛查,并有助于探索微生物的功能基因组学。



表1.定义的E. piscicida EIB202突变体文库的统计值

资料集

数字

映射的插入位点

24470

突变体映射

20668

插入不明确

4124

在ORF中插入

18128

ORF之间的插入

6342

在5-85%ORF内插入

14028

5-85%ORF以外的插入

4100

转录融合

12295

转座子-插入框架

12383

转座子+框架插入

12087

EIB202上的带注释的ORF

3599

ORF中断

2806

ORF永不中断

793

每个带注释的ORF的平均点击数

5.04

预测的必需基因

464

16 °C海水中的条件必需基因

158

28 °C海水中的条件必需基因

75

DMEM培养基中的条件必需基因

52

J774A.1中的条件必需基因

67

大菱t中的条件必需基因

258





图1.定义的转座子突变体文库构建。A. pMKGR ,的衍生物Himar1水手转座子,构建构建的定义转座子突变体文库E. piscicida EIB202 。B.定义的突变体库构建的工作流程。大肠杆菌SM10被用作一个用于与受体菌株接合供体E. piscicida EIB202。在30 ℃下,在LB板上的EIB202和SM10之间进行偶联处理3 h 。然后,在含有20 µg / ml多粘菌素和15 µg / ml庆大霉素的LB平板上分离偶联物。在30 °C下孵育24小时后,将单克隆抗体放入96孔板中,并在30 °C下孵育大约16小时。前存储于-80 ℃下,丙三醇的溶液中加入到每个孔,并混合以制备最终20%甘油浓度保持株。C.用于确定基因组上的Tn插入基因座的TAIL-PCR扩增的示意图。一对引物SP1 / AB2的用于第一轮PCR的另一对的引物的Sp2 / ABS用于在第二轮PCR。最后,将引物Seq2用于扩增的PCR产物的测序。D.新破坏基因的数量随着新突变体的增加而线性增加,命中的基因数量接近平稳状态。

关键字:转座子插入测序, 确定突变体文库测序, 确定突变体库, 条件性必需基因

材料和试剂


处理细菌
大方培养皿(500 cm 2 )(Thermo Fisher Scientific ,目录号:R80115TS )
纤维素过滤器膜(0.45微米孔径,默克)
Eppendorf管(Axygen ,目录号:MCT-150-C)
PCR管(Axygen ,目录号:PCR-02-A )             
康宁50毫升离心管(默克(Merck),目录号:CLS430828-500EA )
96孔深孔板(Thermo Fisher Scientific ,目录号:A43075)
冷冻管(Thermo Fisher Scientific ,目录号:5000-1020 )
野生型E. piscicida EIB202(山口- [R ); “收件人”单元格
大肠杆菌SM10 λ PIR窝藏pMKGR用于经由接合(提供Tn的安培- [R ,公里- [R ); “供体”细胞(参见注释1 )
Luria-Bertani(米勒的LB汤底,Thermo Fisher Scientific ,目录号:12995027 )和琼脂培养基(默克公司,目录号:05040 )
Colistin(Col,20毫克/毫升,BBI)
氨苄西林(Amp,100 mg / ml,BBI)
庆大霉素(Gm,15 mg / ml,BBI)
甘油(Titan ,目录号:56-81-5 )


热不对称交错PCR(TAIL-PCR)
Tiangen 2 × Taq PCR MasterMix (TIANGEN BIOTECH ,目录号:KT205-02)
无核酸酶的水H 2 O(QIAGEN ,目录号:129114)


基因组DNA(gDNA)纯化
0.5 ml单管(Thermo Fisher Scientific,目录号:AB0350)
Tiangen gDNA提取试剂盒(TIANGEN BIOTECH ,目录号:DP302-02)
100%乙醇(Titan,目录号:64-17-5 )


图书馆建设
Qiagen凝胶纯化试剂盒(QIAGEN,目录号:28104)
VAHTS HiFi扩增混合液(Vazyme Biotech ,目录号:616-01 / 02)
琼脂糖(Thermo Fisher Scientific,目录号:16500500)和电泳仪(BIO-RAD ,目录号:1704489EDU)
Qubit TM dsDNA HS检测试剂盒(Invitrogen ,目录号:Q32851)


寡核苷酸DNA (参见注释2 )(HPLC纯化,BGI)
“ EIB202-F”:5'-TCATCGCACATACAGAATAAACGCC-3'
“ EIB202-R”:5'-CCGTAACATTTCTTACAACACTGCG-3'
“ pMKGR -F”:5'-AGGTGATGCTACATACGGAAAG-3'
“ pMKGR -R”:5'-AGCGCATGAACTCCTTGATG-3'
“ Tn-F”:5'-AAAAGTCCGCTGGCAAAG-3'
“ Tn-R”:5'-CCCTTCAAGAGCGATACAAC-3'
“ Sp1-F”:5'-GCTCCGTAGTAAGACATTCATCGCG-3'
“ Sp2-F”:5'-GCTTACGTTCTGCCCAAGTTTGAG-3'
“ ABS-R”:5'-GGCCACGCGTCGACTAGTAC-3'
“ AB2-R”:5'-GGCCACGCGTCGACTAGTACNNNNNNNNNCTCT-3'
“ Seq2-F”:5'-CAATTCGTTCAAGCCGAGATCG-3'
“ AD_fork截短的NH2”:5'- TACCACGACCA-3'
“ AD_Index Fork R”:5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATGGTCGTGGT-3'
“ 1st_seq-out-psc189”:5'-ACTCTGGGGTACGCGTCTAG-3'
“ 1st_PCR_Index'R'引物”:5'-GTGACTGGAGTTCAGACGTGTG-3'
“ P5间隔引物”:以下6个寡核苷酸的等摩尔混合物:
5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGACTTATCAGCCAACCTGT-3'


5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTCGACTTATCAGCCAACCTGT-3'


5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTATGACTTATCAGCCAACCTGT-3'


5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTGTCGACTTATCAGCCAACCTGT-3'


5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTTCGACGACTTATCAGCCAACCTGT-3'


5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTGCAGCGACGACTTATCAGCCAACCTGT-3'


“ P7条码引物”
CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC(因样品而异)






Ë quipment


多通道移液器(Thermo Fisher Scientific)
高速离心机(Eppendorf ,型号:5415R)
热循环仪(E ASTWIN,型号:ETC811)
水浴(Thermo Fisher Scientific ,目录号:TSGP02 )
高速离心机(Eppendorf ,型号:5415R)
浴超声仪(Diagenode ,目录号:B01020001)
热循环仪(E ASTWIN,目录号:ETC811)
VAHTS用于Illumina的通用端制备模块(Vazyme Biotech ,目录号:N203-01)
用于Illumina的VAHTS通用适配器连接模块(Vazyme Biotech ,目录号:N204-01)
Qubit TM 4荧光计(Invitrogen,目录号:Q33238)或等效的基于荧光的DNA定量设备


程序


家长图书馆建设
接种“供体”(在含Amp的LB肉汤中,100 μ克/ ml)和“受体”的细胞(在LB肉汤与山口,20 μ克/毫升),在37 ℃振荡和培养过夜。
准备不含抗生素的LB琼脂平板。让他们干井和地点0.45微米在板的顶部的膜过滤器(4层膜可以施加到每个板)(见注3 )。
通道E. piscicida ,将2ml在50ml(4%)中,添加氯化钙2至50毫米(500 μ升,10 M),在30旋转200转°C,培养1 h 15分钟后OD 600将达到〜0.6-0.8(请参见注释4 )。
测量SM10和piscicida的所有细菌的OD 600 ,预计分别为0.8 。
将培养物移入50 ml康宁管中,在10 °C下旋转5,000 × g 5分钟,然后用10 ml LB洗涤,然后在5,000 × g旋转10 min。
重悬于LB与所述下面的体积以达到最终OD 600 = 4。
混合SM10和等体积E. piscicida在一个Eppendorf管,并且点4 * 80 μ升在步骤A2中制备的过滤器。让drop吸附到琼脂中并干燥。
将这些板放入30 °C的培养箱中,注意不要使细胞悬液溢出到膜外(请勿翻转板)。
孵育3小时后,除去过滤器进入一个由镊子50毫升离心管中。通过上下移液和涡旋使过滤器中的细胞重悬在1 ml的LB肉汤中。
将重悬细胞的系列稀释液进行稀释,然后将100 µl 1:1、1:10和1:100稀释液置于含有Col和Gm的LB琼脂板上(以估计转座子的插入效率,因为实际的文库板也可能变得如此)拥挤地挑选殖民地)。在30 °C下孵育过夜。
检查〜100山口ř了Gm ř通过菌落PCR菌落3对通用引物EIB202-F / R,pMKGR -F / R ,和Tn的-F / R(见注5 )。
将单克隆抗体放入96个深孔板中,在30 °C下旋转200 rpm过夜。
将900 µl细胞与300 µl 80%甘油混合到冷冻管中,以在-80 °C下存储可回收的文库。


热不对称交错PCR(TAIL-PCR)
对于每种单克隆抗体,请在PCR管中准备第一个反应系统:
0.5 µl细菌培养物(来自小标题3.1)。


1 µl 10 µM“ Sp1-F”。


1 µl 10 µM“ AB2-R”。


10 µl Tiangen 2 × Taq PCR MasterMix 。


将H 2 O加入20 µl。


在该程序下的PCR Thermo Cycler上运行。
1 = 94°C持续4分钟。


2 = 94°C持续30 s。


3 = 42°C持续30 s。


[选项]每个周期-1°C。


4 = 72°C,持续3分钟。


5 =转到2,共6次。


6 = 94°C持续30 s。


7 = 58°C持续30 s。


8 = 72°C 1分钟。


9 =转到6,共25次。


10 = 72°C,持续3分钟。


将PCR产物稀释十倍,在PCR管中准备第二个反应系统:
1 µl稀释PCR产物。


2 µl 10 µM“ Sp2-F”。


2 µl 10 µM“ ABS-R”。


25 µl Tiangen 2 × Taq PCR MasterMix 。


将H 2 O加入50 µl。


在该程序下的PCR Thermo Cycler上运行。
1 = 94°C持续3分钟。


2 = 94°C持续30 s。


3 = 64°C持续30 s。


4 = 72°C 1分钟。


5 =转到2,共30次。


6 = 72°C,持续3分钟。


用引物“ Seq2”收集PCR产物和序列。


N冗余子集库的构建(请参阅注释6 ,以E. piscicida EIB202为例)
序列结果被映射到受体EIB202基因组。
绘制饱和度曲线以确保库的饱和度。
根据识别的插入位点,进行了手动生成的非冗余子集库,以促进一个全基因组筛选。
根据不同的实验目的,非冗余的子集库将用于在屏幕上。


gDNA提取
使用市售的gDNA提取试剂盒,请参阅相关协议。


超声处理gDNA剪切
在新鲜的0.5 ml单个试管中稀释gDNA,使其在100 µl H 2 O中的终浓度为50 ng / µl 。
对DNA进行超声处理以产生片段:30s开/ 90s关,持续12个循环。
在琼脂糖凝胶(2%)上运行消化液样品(5 µl)。大多数DNA应该落在200-500 bp的范围内,这对于下游连接和PCR来说是合适的。


结束维修和计费
在PCR管中准备反应:
VAHTS Turbo End Prep酶混合物:3.0 µl。


VAHTS涡轮末端制备反应缓冲液(10 × ):6.5 µl。


片段DNA(50 ng / µl,加1 µg):20 µl。


将H 2 O加入65.0 µl。


使用以下循环条件进行PCR。
1 = 20°C持续30分钟。


2 = 65°C,持续30分钟。


适配器连接
在PCR管中准备以下适配器混合物。
5 µl 100 µM“ AD_fork截短的NH2”。


5 µl 100 µM“ AD_Index货叉R”。


0.4 µl 2 mM氯化镁2。


在该程序下的PCR Thermo Cycler上运行。
1 = 95°C持续4分钟。


2 = 95°C 1分钟。


[选项]每个周期-1°C。


3 =转到2,进行75次。


4 =结束。


稀到一半浓度:添加了H的相同体积的2 O.
在PCR管中准备以下连接反应。
A-尾DNA(来自上述小标题3.5):65微升。


VAHTS Turbo T4 DNA连接酶:2.0 µl。


VAHTS Turbo Ligation Enhancer:30.5 µl。


Tn-seq的适配器(从小标题3.6,第2步开始):2.5 µl。


将H 2 O加入100 µl。


将反应在20°C下孵育30分钟。
柱纯化的DNA,用35 µl预热的H 2 O洗脱。


Tn相关gDNA的扩增
制备在以下的PCR反应的Eppendorf管中。
50 µl VAHTS HiFi扩增混合物。


200 ng连接的DNA(来自小标题3.6)。


5 µl 10 µM“ 1st_seq-out-psc189引物”。


5 µl 10 µM“ 1st_PCR_Index'R'引物”。


将H 2 O加入100 µl。


分装到2个PCR管中(每个50 µl),并在以下循环条件下进行PCR。
1 = 98°C持续30 s。


2 = 98°C持续10 s。


3 = 53°C持续30 s。


4 = 72°C持续30 s。


5 =转到2,共29次。


6 = 72°C,持续5分钟。


合并2个PCR反应,然后用35 µl预热的H 2 O洗脱纯化PCR产物。


第二次PCR以添加条形码,Illumina附件(P5,P7)和可变性序列
制备在以下的PCR反应的Eppendorf管中。
300 ng纯化的PCR产物(来自小标题3.7)。


75 µl VAHTS HiFi扩增混合物。


7.5 µl 10 µM“ P5间隔引物”。


7.5 µl 10 µM P7 Index条码(应随样品的不同而不同)。


将H 2 O加入150 µl。


分装到3个PCR管中(每个50 µl),并在以下循环条件下进行PCR。
1 = 98°C持续30 s。


2 = 98°C持续10 s。


3 = 55°C持续30 s。


4 = 72°C持续30 s。


5 =转到2,共17次。


6 = 72°C,持续7分钟。


合并3个PCR反应,然后用35 µl预热的H 2 O洗脱纯化PCR产物。


尺寸选择
在0.5 × TAE缓冲液中的2%琼脂糖凝胶上运行所有纯化的PCR产物(来自小标题3.8)。
通过剪切200至500 bp之间的拖尾来选择大小。请确保不包括约200基点可见头对头引物二聚体。
使用标准凝胶提取程序对DNA进行柱纯化,并用50 µl预热的H 2 O洗脱。
通过Qubit定量DNA浓度。现在,DNA已准备好在Illumina测序仪上运行。


条件必需基因分析
使用EL-ARTIST管道确定转座位点和插入的丰度(Chao等,2013)。基于所述中性碱基对模型,转座子插入的随机分布,含有库Ñ突变体,并且在总的基因数目的基因组是ķ ,通过转座子揭露的基因的数量是N.基因j的插入得到的概率p j ,转座子的未发现基因数为N t = 。将必需基因数设为k 1。作为中性碱基对模型,未发现必需基因的概率为N e =   ,并且预测的必需基因数k 1 = N- N t + N e 。


笔记


pMKGR是基于Himar1 Mariner转座子构建的。扩增了无启动子的卡那霉素抗性(Kan r )盒和具有核糖体结合位点(RBS)的基因egfp 。在这项研究中使用的RBS序列是“ AAGGAGG”,其源自大肠杆菌16S rRNA 3'末端保守序列“ CCUCCUU”。将三重末端位点“ TGACTAGCTAA”和48-bp T7终止子序列引入到pMKGR中。将构建的pMKGR转化到大肠杆菌SM10中,并通过PCR和测序进行验证。
的p MKGR ,其衍生物的Himar1水手转座子和菌株E. piscicida EIB202被用作在该协议的例子; 对于不同的转座子和菌株,所有引物序列都需要进行相应的修饰。
制备的膜应与缀合反应一样多。随着越来越多的采摘菌落用于测序,被破坏的基因数量正在增加,并且增加的速率逐渐变为水平,并在步骤C2中达到平稳。这意味着已经产生了大量诱变,并且收集到的突变体能够组装一个全面而饱和的文库。
大肠杆菌EIB202的最佳生长温度为30 °C 。需要根据不同的受体菌株修改以下孵育温度。
当抗生素被用作选择性标记物,有一个小的机会来观察菌落不是真正抗性和能在抗生素存在即使不含有转座子插入(表型抗性)生长。为了确保成功的换位,建议随机restreak 〜100个菌落在新鲜平板并用3对通用引物,其被设计为转座子,其载体质粒和“收件人”小区执行菌落PCR。对于菌落PCR,1)用无菌的扁平牙签或移液器吸头挑选单个菌落,并在无菌水中涡旋。2)大号YSE细菌通过或者简单煮沸使用之前样品或直接加入样品以PCR反应的小体积,以释放DNA。细菌将在常规PCR程序的初始加热步骤中裂解。3)循环条件为:98°C,1分钟;随后进行30个98°C的循环,持续5 s;55°C,5 s; 和72°C,40 s。4)PCR产物的大小是通过电泳确定,然后PCR产物用桑格测序验证。
从亲本库中分离出五个子库。1日和平行2个第二子集文库分别包括单突变为每个ORF,和插入位点在端分别给予优先各ORF-3' 5'内0.4-0.6百分比。一旦ORF仅包含一个插入位点,这些突变体被选择之前到1日的子集库。的3次子集库是一个转录融合文库,其中每个突变体携带MKGR与当地ORF相同的取向和优先考虑接近插入位点的各ORF的5'端。类似于1第一子集库的标准,在4个中生成的库,并且包括不超过两个突变体针对每个基因间区域。5个化合物库是所有从突变体的混合物的1日,2次和4个的子集库和需要2×10 7 CFU为每个相应的突变。


致谢


所描述的协议改编自Wei等人。,2019.这项工作得到了中国国家自然科学基金(32002436授予SS,82010033授予LW),中国国家重点研究与发展计划(2018YFD0900504授予QW),上海航海计划(20YF1411100)的资助。中国农业部科学基金(2020M671034)(CARS-47)和上海市科学技术委员会(17391902000)。




利益争夺


没有利益冲突或利益冲突。


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引用:Shao, S., Wei, L., Xia, F., Zhang, Y. and Wang, Q. (2021). Defined Mutant Library Sequencing (DML-Seq) for Identification of Conditional Essential Genes. Bio-protocol 11(5): e3943. DOI: 10.21769/BioProtoc.3943.
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