A modular chromosomally integrated toolkit for ectopic gene expression in Vibrio cholerae

The ability to express genes ectopically in bacteria is essential for diverse academic and industrial applications. Two major considerations when utilizing regulated promoter systems for ectopic gene expression are (1) the ability to titrate gene expression by addition of an exogenous inducer and (2) the leakiness of the promoter element in the absence of the inducer. Here, we describe a modular chromosomally integrated platform for ectopic gene expression in Vibrio cholerae. We compare the broadly used promoter elements Ptac and PBAD to versions that have an additional theophylline-responsive riboswitch (Ptac-riboswitch and PBAD-riboswitch). These constructs all exhibited unimodal titratable induction of gene expression, however, max induction varied with Ptac > PBAD > PBAD-riboswitch > Ptac-riboswitch. We also developed a sensitive reporter system to quantify promoter leakiness and show that leakiness for Ptac > Ptac-riboswitch > PBAD; while the newly developed PBAD-riboswitch exhibited no detectable leakiness. We demonstrate the utility of the tightly inducible PBAD-riboswitch construct using the dynamic activity of type IV competence pili in V. cholerae as a model system. The modular chromosomally integrated toolkit for ectopic gene expression described here should be valuable for the genetic study of V. cholerae and could be adapted for use in other species.


Fig. S1 -Details on how to assemble the ectopic expression constructs and swap out genes of interest.
A generic diagram of the ectopic expression construct is shown with the location of different primers needed to initially construct each expression construct (P1, P2, P3, P6, P7, and P8). As well as the primers to insert novel genes of interest into established expression constructs (P1, P4, P5, P6, P7, and P8). Ab R = antibiotic resistance cassette, reg = regulatory gene (lacIq or araC), P X = the promoter (P tac , P BAD , P tac -riboswitch, or P BADriboswitch), and gene X = the gene of interest.

Establishing ectopic expression constructs at new genomic locations:
The UP arm of homology is generated with P1 and P2, while the DOWN arm of homology is amplified with P7 and P8. In this example, these primers amplify homology arms to integrate the ectopic expression constructs into V. cholerae ChII. P2 and P7 contain tails that overlap any of the 4 ectopic expression constructs. To move intact expression constructs to new locations in the genome, they can be amplified with P3 and P6. This can serve as a MIDDLE arm to stitch to the UP and DOWN arms of homology.

To insert new genes of interest:
Once an ectopic expression construct has been introduced to a genetic locus, the gene of interest can be swapped out.

Fig. S1 -Details on how to assemble the ectopic expression constructs and swap out genes of interest
A generic diagram of the ectopic expression construct is shown with the location of different primers needed to initially construct each expression construct (P1, P2, P3, P6, P7, and P8). As well as the primers to insert novel genes of interest into established expression constructs (P1, P4, P5, P6, P7, and P8). Ab R = antibiotic resistance cassette, reg = regulatory gene (lacIq or araC), PX = the promoter (Ptac, PBAD, Ptac-riboswitch, or PBAD-riboswitch), and gene X = the gene of interest.
Establishing ectopic expression constructs at new genomic locations: The UP arm of homology is generated with P1 and P2, while the DOWN arm of homology is amplified with P7 and P8. In this example, these primers amplify homology arms to integrate the ectopic expression constructs into V. cholerae ChII. P2 and P7 contain tails that overlap any of the 4 ectopic expression constructs. To move intact expression constructs to new locations in the genome, they can be amplified with P3 and P6. This can serve as a MIDDLE arm to stitch to the UP and DOWN arms of homology.
To insert new genes of interest: Once an ectopic expression construct has been introduced to a genetic locus, the gene of interest can be swapped out.
-To amplify a gene of interest to place within Ptac-riboswitch or PBAD-riboswitch the forward P5-1 primer must have a 5' overlap region as indicated. Immediately after this overlap should be the start codon for the gene of interest (highlighted in green) + additional sequence to serve as a primer for the gene of interest (denoted by 'XXXXXX'). P5-1 overlaps with P4-1 (blue highlighted sequence), which sits within the theophylline-dependent riboswitch. Thus, the same overlap can be used to make either Ptac-riboswitch or PBAD-riboswitch constructs. The reverse P6 primer for the gene of interest must have the tail indicated, which should be immediately followed by the stop codon of the gene (highlighted in purple -TAA stop codon in this example) + additional sequence to serve as a primer for the gene of interest (denoted by 'YYYYYY').
-To amplify a gene of interest to place within Ptac or PBAD the forward P5-2 primer must have the 5' overlap region indicated. Immediately after this overlap should be the desired ribosome binding site (indicated in bold and underline -the 'optimal' RBS AGGAGGT is used in this example), which should be followed by a 6 bp spacer (this spacing between the ribosome binding site and the start codon is essential for optimal translation and can be derived from the native gene) + the start codon (highlighted in green) + additional sequence to serve as a primer for the gene of interest (denoted by 'XXXXXX'). P5-2 overlaps with P4-2 (blue highlighted sequence), which sits downstream of the two promoter elements. Thus, the same overlap can be used to make either Ptac or PBAD constructs. The reverse P6 primer for the gene of interest can be made exactly as described above.
All amplified genes of interest serve as MIDDLE arms in SOE reactions with an UP arm amplified with P1 and P4, and a DOWN arm amplified with P7 and P8.
-To amplify a gene of interest to place within P tac -riboswitch or P BAD -riboswitch the forward P5-1 primer must have a 5' overlap region as indicated. Immediately after this overlap should be the start codon for the gene of interest (highlighted in green) + additional sequence to serve as a primer for the gene of interest (denoted by 'XXXXXX'). P5-1 overlaps with P4-1 (blue highlighted sequence), which sits within the theophylline-dependent riboswitch. Thus, the same overlap can be used to make either P tac -riboswitch or P BAD -riboswitch constructs. The reverse P6 primer for the gene of interest must have the tail indicated, which should be immediately followed by the stop codon of the gene (highlighted in purple -TAA stop codon in this example) + additional sequence to serve as a primer for the gene of interest (denoted by 'YYYYYY').
-To amplify a gene of interest to place within P tac or P BAD the forward P5-2 primer must have the 5' overlap region indicated. Immediately after this overlap should be the desired ribosome binding site (indicated in bold and underline -the 'optimal' RBS AGGAGGT is used in this example), which should be followed by a 6 bp spacer (this spacing between the ribosome binding site and the start codon is essential for optimal translation and can be derived from the native gene) + the start codon (highlighted in green) + additional sequence to serve as a primer for the gene of interest (denoted by 'XXXXXX'). P5-2 overlaps with P4-2 (blue highlighted sequence), which sits downstream of the two promoter elements. Thus, the same overlap can be used to make either P tac or P BAD constructs. The reverse P6 primer for the gene of interest can be made exactly as described above.
All amplified genes of interest serve as MIDDLE arms in SOE reactions with an UP arm amplified with P1 and P4, and a DOWN arm amplified with P7 and P8.  Fig. S1)