Materials and Reagents

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 (Col^R); “recipient” cells
9. E. coli SM10 λpir harboring pMKGR for delivering Tn via conjugation (Amp^R, Km^R); “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 )
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 )
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 )
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. Qubit^TM dsDNA HS Assay kit (Invitrogen, catalog number: Q32851 )
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

Multichannel pipette (Thermo Fisher Scientific)
High speed centrifuge (Eppendorf, model: 5415R )
Thermal cycler (EASTWIN, model: ETC811 )
Water bath (Thermo Fisher Scientific, catalog number: TSGP02 )
High speed centrifuge (Eppendorf, model: 5415R )
Bath sonicator (Diagenode, catalog number: B01020001 )
Thermal cycler (EASTWIN, catalog number: ETC811 )
VAHTS Universal End preparation Module for Illumina (Vazyme Biotech, catalog number: N203-01 )
VAHTS Universal Adapter Ligation Module for Illumina (Vazyme Biotech, catalog number: N204-01 )
Qubit^TM 4 Fluorometer (Invitrogen, catalog number: Q33238 ), or equivalent fluorescent-based DNA quantification apparatus

Procedure

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 Col^R Gm^R 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.

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”.

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.

gDNA Extraction
Use commercial gDNA extraction kit, refer to relevant protocol.

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.

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.

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 MgCl_2.
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.

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.

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.

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.

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 p_j, 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 - N_t + N_e.

Notes

pMKGR was constructed based on Himar1 mariner transposon. The promoterless kanamycin resistance (Kan^r) 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.
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.
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.
The optimal growth temperature of E.piscicida EIB202 is 30 °C. The following incubation temperature needs to be modified according to different recipient strains.
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.
Five subset libraries were separated from the parental library. The 1^st and parallel 2^nd 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 1^st subset library. The 3^rd 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 1^st subset library criteria, the 4^th library was generated and includes no more than two mutants for each intergenic region. The 5^th compound library is the mixture of all the mutants from the 1^st, 2^nd and 4^th subset libraries and takes 2 x 10⁷CFU 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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