TABLE 3.
Primers used in this study
| Primer | Sequence (5′→3′) | Description |
|---|---|---|
| pDS132-hifi-f | CGATCCTTTTTAACCCATCAC | Forward primer to amplify pDS132 vector |
| pDS132-hifi-r | CTTCTAGAGGTACCGCATGC | Reverse primer to amplify pDS132 vector |
| pDS132-f | TGTTGCATGGGCATAAAGTTGC | Forward confirmation primer of pDS132 vector |
| pDS132-r | ACGGCTGACATGGGAATTCC | Reverse confirmation primer of pDS132 vector |
| pDS132-VipA-sfGFP KI-1 | AAAAGGATCGATCCTCTAGAGAGTGCGTGGAGGATATGGG | Forward primer to amplify upstream to construct insertion of sfGFP |
| pDS132-VipA-sfGFP KI-2 | TCCTCCTCCTGCGGCCGCGGCCTCGGCAGGCTTAATCAAA | Reverse primer to amplify upstream to construct insertion of sfGFP |
| pDS132-VipA-sfGFP KI-3 | TTTGATTAAGCCTGCCGAGGCCGCGGCCGCAGGAGGAGGA | Forward primer to amplify gene of sfGFP |
| pDS132-VipA-sfGFP KI-4 | AGTCAATCCAACCCGGCGATTTATTTGTAGAGCTCATCCA | Reverse primer to amplify gene of sfGFP |
| pDS132-VipA-sfGFP KI-5 | TGGATGAGCTCTACAAATAAATCGCCGGGTTGGATTGACT | Forward primer to amplify downstream to construct insertion of sfGFP |
| pDS132-VipA-sfGFP KI-6 | TCGCATGCGGTACCTCTAGAACCAGCGATACTTGGCGAAA | Reverse primer to amplify downstream to construct insertion of sfGFP |
| pDS132-VipA-N3-sfGFP KI-1 | GTGATGGGTTAAAAAGGATCGGCATGGTATCTGCTTGCTGC | Forward primer to amplify upstream to construct insertion of N3 |
| pDS132-VipA-N3-sfGFP KI-2 | CTCTATTTCCGCAACCTCAGCCTGCTGACCACCGGTTGCCGGTA | Reverse primer to amplify upstream to construct insertion of N3 |
| pDS132-VipA-N3-sfGFP KI-3 | GCTGAGGTTGCGGAAATAGAGCTTCCGTTGA | Forward primer to amplify downstream to construct insertion of N3 |
| pDS132-VipA-N3-sfGFP KI-4 | GCATGCGGTACCTCTAGAAGGATAGTGGTCGTGATCCGAGCT | Reverse primer to amplify downstream to construct insertion of N3 |
| pDS132-VipA-N3-sfGFP KI-5 | GGTATGATGCGAGAGCAGGG | Forward confirmation primer of N3 mutation |
| pDS132-VipA-N3-sfGFP KI-6 | ACCAGCGATACTTGGCGAAA | Reverse confirmation primer of N3 mutation |
| pDS132-VipA-N3-sfGFP KI-confirm | ACGCATCGTCAGCTGATGAACTA | Confirmation primer for sequencing of N3 insertion |
| pDS132-B-NheI-KI-1 | GTGATGGGTTAAAAAGGATCGGGGAAGTAAAAACGGCGATG | Forward primer to amplify upstream of back NheI site to construct insertion of NheI |
| pDS132-B-NheI-KI-2 | CAAACTCGCTAGCTTAACCATATCTTTCTTTCATATACTTCTTTACC | Reverse primer to amplify upstream of back NheI site to construct insertion of NheI |
| pDS132-B-NheI-KI-3 | TGGTTAAGCTAGCGAGTTTGGCAAGGTGTGATAGAC | Forward primer to amplify downstream of back NheI site to construct insertion of NheI |
| pDS132-B-NheI-KI-4 | GCATGCGGTACCTCTAGAAGGCCCTTAGCTCAGTCGGATAG | Reverse primer to amplify downstream of back NheI site to construct insertion of NheI |
| pDS132-B-NheI-KI-5 | ATTATGCATTCAATCCTGTGGC | Forward confirmation primer of insertion of back NheI site |
| pDS132-B-NheI-KI-6 | GCGCAACCTGTTTTCCATCA | Reverse confirmation primer of insertion of back NheI site |
| pDS132-B-NheI-KI-confirm | GCAGAAGGGATTGATGAGACA | Confirmation primer for sequencing of insertion of back NheI site |
| PDS132-F-KI-NheI-NP-KI-1 | GTGATGGGTTAAAAAGGATCGCAAAAAGAAGCGAGGGCC | Forward primer to amplify upstream of front NP-NheI site to construct insertion of NheI |
| PDS132-F-KI-NheI-NP-KI-2 | CCACTCGGCTAGCGACACCTCACCAAATTTTTACTTCTTG | Reverse primer to amplify upstream of front NP-NheI site to construct insertion of NheI |
| PDS132-F-KI-NheI-NP-KI-3 | AGGTGTCGCTAGCCGAGTGGCTGAAGGAGCAC | Forward primer to amplify downstream of front NP-NheI site to construct insertion of NheI |
| PDS132-F-KI-NheI-NP-KI-4 | GCATGCGGTACCTCTAGAAGCTTGCACCAGCATCTCGTCT | Reverse primer to amplify downstream of front NP-NheI site to construct insertion of NheI |
| PDS132-F-KI-NheI-NP-KI-5 | TGGATGGTCATTTAGCCCCC | Forward confirmation primer of insertion of front NP-NheI site |
| PDS132-F-KI-NheI-NP-KI-6 | AGCAGGCTGACCAGATTGC | Reverse confirmation primer of insertion of front NP-NheI site |
| PDS132-F-KI-NheI-NP-KI-confirm | TAAATTACGCCCCGTTCCAGC | Confirmation primer for sequencing of insertion of front NP-NheI site |
| PDS132-F-KI-NheI-RBS-KI-1 | GTGATGGGTTAAAAAGGATCGGCGTTAAATGTGCGAACGGT | Forward primer to amplify upstream of front RBS-NheI site to construct insertion of NheI |
| PDS132-F-KI-NheI-RBS-KI-2 | TGCTCCTGCTAGCGATTGGTTGAACGGTAATGACACT | Reverse primer to amplify upstream of front RBS-NheI site to construct insertion of NheI |
| PDS132-F-KI-NheI-RBS-KI-3 | ACCAATCGCTAGCAGGAGCAATTCCATGCCAAC | Forward primer to amplify downstream of front RBS-NheI site to construct insertion of NheI |
| PDS132-F-KI-NheI-RBS-KI-4 | GCATGCGGTACCTCTAGAAGGAAGGTGTTTTCGGGCAGAG | Reverse primer to amplify downstream of front RBS-NheI site to construct insertion of NheI |
| PDS132-F-KI-NheI-RBS-KI-5 | CGACCCATGAAGTGGTCTCC | Forward confirmation primer of insertion of front RBS-NheI site |
| PDS132-F-KI-NheI-RBS-KI-6 | GCGCGAAGTCACTCACCACC | Reverse confirmation primer of insertion of front RBS-NheI site |
| PDS132-F-KI-NheI-RBS-KI-confirm | TTTCCTATTGTCAACGCATAGCTCA | Confirmation primer for sequencing of insertion of front RBS-NheI site |
| pDS132-tsePE663A-hifi-f | GTGATGGGTTAAAAAGGATCGCGGTGAGGTGGTTGGACATTG | Forward primer to amplify upstream and downstream of tsepE663 to construct mutation of tsepE663A |
| pDS132-tsePE663A-hifi-r | GCATGCGGTACCTCTAGAAGCGCTCCTGAGATTCGGGTAATC | Reverse primer to amplify upstream and downstream of tsepE663 to construct mutation of tsepE663A |
| tsePE663A-f | GCCAAAGATATGTTGGGTCAACC | Forward confirmation primer of tsePE663A mutation |
| tsePE663A-r | CAGTATATTCTCCACCAGCGTTCTTTTT | Reverse confirmation primer of tsePE663A mutation |
| tsePE663A-confirm | CTTATCACTCTAGAAATGGTTTTTGCAGC | Confirmation primer for sequencing of tsePE663A mutation |
| pDS132-tseIHFH-AAA-hifi-f | GTGATGGGTTAAAAAGGATCGCGAGCGCTTCTTGCATGATCC | Forward primer to amplify upstream and downstream of tseIHFH to construct mutation of tseIHFH-AAA |
| pDS132-tseIHFH-AAA-hifi-r | GCATGCGGTACCTCTAGAAGACGATATGCCTCTTCAAAAAACATCCT | Reverse primer to amplify upstream and downstream of tseIHFH to construct mutation of tseIHFH-AAA |
| tseIHFH-AAA-f | CTATGGCTATGACAGAACGGGCA | Forward confirmation primer of tseIHFH-AAA mutation |
| tseIHFH-AAA-r | CACTACCTTGTAATAAGACCAACGACCA | Reverse confirmation primer of tseIHFH-AAA mutation |
| tseIHFH-AAA-confirm | GCGCAATATAAAACACATAAATATGTTTATG | Confirmation primer for sequencing of tseIHFH-AAA mutation |
| pDS132-vasK-ko1 | GTGATGGGTTAAAAAGGATCGTCTAGACCATGGTACTCAGCGGTGG | Forward primer to amplify upstream and downstream of vasK to construct deletion of vasK |
| pDS132-vasK-ko4 | GCATGCGGTACCTCTAGAAGTCTAGATCTCCTGCTGATGTTGCTGC | Reverse primer to amplify upstream and downstream of vasK to construct deletion of vasK |
| pDS132-vasK-ko5 | GTTGGTGATGGGGCTGAAA | Forward confirmation primer of deletion of vask |
| pDS132-vasK-ko6 | GCGAATGTCGTCGTAGCTGA | Reverse confirmation primer of deletion of vask |
| p15A-Tet-r | GTGAAGGCACCGGCGGTGATGTTGCCCTGAGTTTTACCTTCGATGCTGATATAACATGGAGTTGGCATGGAATTGCTCCTCTTTAGATCTTTTGAATTCTTTTCTCTATC | Reverse primer to amplify p15A-cm-tetR-tetO-hyg-ccdB linear vector with 80-bp homologous arm target to T6SS cluster (without native promoter of hcp1) |
| p15A-f | AGGATGGGGCCGGTGATGGAACATATAAGTTTAACAATGCAATATCGGTAAAGAAGTATATGAAAGAAAGATATGGTTAAAGATCCGAAAACCCCAAGT | Forward primer to amplify p15A-cm-tetR-tetO-hyg-ccdB linear vector with 80-bp homologous arm target to T6SS cluster |
| p15A-NP-r | ATTTGGCGGTGAGGGAGGGATTCGAACCCTCGATACGTTGCCGTATACACACTTTCCAGGCGTGCTCCTTCAGCCACTCGCTTTAGATCTTTTGAATTCTTTTCTCTATC | Reverse primer to amplify p15A-cm-tetR-tetO-hyg-ccdB linear vector with 80-bp homologous arm target to T6SS cluster (with native promoter of hcp1) |
| bla-f | TCGAAATGAGTGCCGAAATGAGTCATTGAGTCGGAGTTGGATCACATTTTATGAGTATTCAACATTTCCGTGTCG | Forward primer to amplify bla with 50-bp homologous arm target to vasK |
| bla-r | ACATATGAACTTCCAACACAGGAAGGGGGCACAACGTGCCCCCGACGCGATTACCAATGCTTAATCAGTGAGGC | Reverse primer to amplify bla with 50-bp homologous arm target to vasK |
| p15A-cm-tetR-tetO-f | AGATCTAAAGAGATCCGAAAACCCCAAGT | Forward primer to amplify p15A empty vector without hyg and ccdB |
| p15A-cm-tetR-tetO-r | TTTCGGATCTCTTTAGATCTTTTGAATTCTTTTCTCTATC | Reverse primer to amplify p15A empty vector without hyg and ccdB |
| ΔvasK-confirm-f | CAAGAGGCCAGTCGCCTC | Forward confirmation primer of deletion of vasK and the insertion of bla |
| ΔvasK-confirm-r | GTACTCCGGCAACATCCTGG | Reverse confirmation primer of deletion of vasK and insertion of bla |
| p15A-confirm-f | GTTAAACCTTCGATTCCGACCT | Forward confirmation primer of sequence of front 80-bp homologous arm in recombinant plasmid |
| p15A-confirm-r | TATAAACGCAGAAAGGCCCAC | Reverse confirmation primer of sequence of back 80-bp homologous arm in recombinant plasmid |
| tsiI-3V5-KpnI-f | CCGGTACCAGGAGGAAACGATGAATTCT | Forward primer to amplify tsiI with 3V5 tag |
| tsiI-3V5-XbaI-r | GCTCTAGATTATTATGTTGAATCAAGTCCGAGAAGTGG | Reverse primer to amplify tsiI with 3V5 tag |
| pPSV37-f | AAGGGTGGAAACGCAAAA | Forward confirmation primer of pPSV37 |
| pPSV37-r | CCCCTCAAGACCCGTTTAGA | Reverse confirmation primer of pPSV37 |
| tseCtsiC-f | TTGGGCTAGCAGGAGGTACCATGAGTACGCCCAATCAAGCC | Forward primer to amplify tseC tsiC |
| tseCtsiC-r | GGTTTACCGCATGCTCTAGATTACTTATTGATCTCATCCAGCCTTCTTG | Reverse primer to amplify tseC tsiC |
| 2404-2401-f | TTGGGCTAGCAGGAGGTACCATGGCAGACAGCACAGGATTACA | Forward primer to amplify genes from 2404 to 2401 |
| pBAD24-KpnI-hifi-R | GGTACCTCCTGCTAGCCCAAAA | Reverse primer to amplify pBAD24 vector |
| pBAD24-KpnI-hifi-F | TCTAGAGCATGCGGTAAACCTATTCC | Forward primer to amplify pBAD24 vector |