Plasmid construction.

All primers are listed in Supplementary Table 3 and plasmids are listed in Supplementary Table 2. All PCR products were amplified using Q5-High Fidelity DNA polymerase according to the manufacturer (New England Biolabs).

pMJF103 was constructed as an arabinose-inducible vector that could be conjugated. pEVS141 vector backbone encoding the kanamycin resistance marker, p15A oriV and oriT was amplified with primers oMJF043 and oMJF044. An arabinose-inducible cassette including araC and mOrange under the pBAD promoter was amplified from the construct pBAD_mOrange (a gift of Patricia Champion) using Q5 and primers oMJF041 and oMJF042. Amplifications were confirmed by gel electrophoresis and treated with DpnI enzyme to remove template. Products were mixed 1:1 and transformed into chemically competent DH5α E. coli and plated on LB agar with 100 μg/mL kanamycin.

Transformants were picked for overnight broth culture in LB supplemented with 100 μg/mL kanamycin and 0.01% L-arabinose. Cultures showing mOrange expression were subsequently miniprepped using a Wizard miniprep kit (Promega). To remove the mOrange insert and generate and empty cloning vector, plasmid was double digested with NdeI and SpeI restriction enzymes (NEB) followed by gel purification of the 4725 bp band using a Wizard spin column (Promega). A multiple cloning site linker was prepared by annealing the primers oMJF055 and oMJF056 at 8 μM each in a buffer of 50 mM Tris HCl pH7.5, 50 mM NaCl and 1 mM EDTA. Linker was ligated into purified linearized vector using T4 ligase (NEB) at 4°C overnight followed by transformation into DH5α E. coli. Presence and orientation of the linker was confirmed by colony PCR using GoTaq polymerase (Promega) with primers oMJF042 and oMJF055. Colonies with confirmed insert were grown overnight in LB broth with 100 μg/mL kanamycin and plasmids were purified by miniprep.

To generate the ptgvAB, tgvAB was amplified from pJBG007 with 520bp upstream sequence to include its native promoter and overlapping ends to the pMJF103 using oJBG122 and oJBG106. To generate ptgvA, tgvA was amplified from pJBG007 with 520bp upstream sequence to include its native promoter and overlapping ends to pMJF103 using oJBG0122 and oJBG108. To generate ptgvB, tgvB was amplified from pJBG029 with 520bp upstream sequence to include its native promoter and overlapping ends to pMJF103 using oJBG122 and oJBG106. To generate T2 pagt, agt was amplified from T2 wild type with overlapping ends to pMJF103 using oJBG137 and oJBG138. To generate T4 pagt, agt was amplified from T4 wild type with overlapping ends to pMJF103 using oJBG133 and oJBG134. To generate T4 pbgt, bgt was amplified from T4 wild type with overlapping ends to pMJF103 using oJBG131 and oJBG132. The pMJF103 backbone was linearized using primers oMJF019 and oMJF053. All PCR products were cleaned and purified using the DNA Clean & Concentrator kit (Zymo Research).

All plasmid constructs were made using fast cloning(⁵³). Amplified inserts and linearized pMJF103 backbone were DpnI treated for at 37°C for 1 hour followed by heat inactivation at 80°C for 20 min. Following DpnI treatment, 1μl of corresponding insert + 1μl of linearized pMJF103 backbone + 50μl of DH5α chemical competent cells were mixed and left on ice for 30 min. After 30 min. tubes were incubated at 42°C for 1 min. and immediately placed on ice for 3 min. 500μl of S.O.C was added, and cells were recovered at 37°C for 1 hour. Cells were spread on LB agar + Kanamycin and left at 37°C overnight. Inserts were verified by PCR amplification of the cloning sight using oMJF057 and oMJF058 primers.

To extract plasmids, Wizard Plus SV Minipreps DNA Purification System kit (Promega) was used following the Centrifugation Protocol under Quick Protocol. Miniprepped plasmids were introduced into DH10β cells by electroporation. All plasmids were confirmed by sequencing (Plasmidsaurus). Plasmid sequencing results were mapped to V. cholerae C6706 Chromosome I ({"type":"entrez-nucleotide","attrs":{"text":"CP064350","term_id":"1932138598","term_text":"CP064350"}}CP064350) or T2 reference genome ({"type":"entrez-nucleotide","attrs":{"text":"NC_054931.1","term_id":"2046350982","term_text":"NC_054931.1"}}NC_054931.1) using Geneious Prime 2023.0.2.