Team:Glasgow/Project/Mobility Proteins

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Motility Genes: MotA and MotB

We were investigating motility genes which would allow bacteria to swim when the switch was off. Our main working strains DS941, DH5α and TOP10 had motA knock-out variants and, thus, we decided to incorporate motA into our switch on a different plasmid and rescue the gene. Wild-type motA was obtained from XB3.1 strain genomic gDNA, as the one already present in the registry didn't have the right beginning (based on Colibri E.coli genome database). Primers were designed to change the starting codon of the gene from GTG to ATG to match registry requirements and stop codon was changed from TGA to a stronger TAA; ribosome binding site was also added.

MotA was then ligated into the pSB1C3 and plasmid J61002 which had a J23100 promoter. Those plasmids were then transformed into strains DH5α and TOP10. First transformation was only successful with the pSB1C3 plasmid whereas transformation of promoter plasmid had to be repeated. Sequencing data shown that pSB1C3 had motA insert without any mutations whereas the only two colonies of J23100 had mutations; in one variant in the ribosome binding site and in another variant in there was a 5 base depletion in the 5' end of the gene. This suggested that promoter J23100 was too strong for motA expression and was possibly toxic to the cells. We decided to use a variant with mutated RBS for our further experiments.
Swarm assays (semi-solid agar motility test) were developed to investigate whether inserted plasmid expressing motA would rescue swimming and also to check whether our knock-outs did not swim. Strain DS941 ΔmotA (with motA knock-out) was transformed with pSB1C3 motA and J23100 motA and then carried out swarm experiments. Strain MG1655-z1 was used as a positive swimming control (Figure 1).

As the first swarm assay didn't show good gene rescuing, we decided to use weaker promoters:

• J23106 (½ the strength of J23100)
• J23116 (¼ the strength of J23100)
• J23103 (very weak promoter)
• J23112 (weakest promoter we could find in the registry, barely no expression)

(Strength measured with RFP: Part BBa_J23100

These promoters were transformed into DS941 and motA ligated downstream of the promoter and then sequenced. After confirmation that all plasmids were correct they were then transformed into DS941 ΔmotA strain and swimming experiment was repeated (Illustration 2).

Figure 2: Illustration 2: Swarm assay. 5µ drop of overnight culture was added on a soft-agar plate and left incubated overnight at 37°C.

Figure 1: Swarm assay. 5µ drop of overnight culture was added on a soft-agar plate and left incubated overnight at 37°C.

Swimming assays using other promoters didn't show gene rescue either. As we were aware of motA-motB gene operon we hypothesized that knock-out of the motA might interfere with expression of motB and, thus, we decided to incorporate motB gene next to the motA.

MotB was then acquired from XB3.1 strain by PCR. Reverse primer had stop codon changed from TGA to a stronger TAA and ribosome binding site added. Promoter plasmids with motA then were digested, motB ligated in and transformed into DS941. Unfortunately, J23100 didn't give any colonies supporting our guess that this promoter is too strong and expression of motility proteins in healthy cells could be toxic. DS941 ΔmotA was then transformed with J23103 motA motB, J23106 motA motB, J23112 motA motB and J23116 motA motB. Gene rescue was checked again by doing swarm assay (Illustration 3). This time we saw a significantly better result than just with motA, however, our parallel investigation on fliC motility genes has shown a better gene rescue.
The distance swam is also correlating with the strength of the promoters. Please see Illustrations 4-6 for distance swam graphs of three swarm assays presented.

Figure 3:Swarm assay. 5µ drop of overnight culture was added on a soft-agar plate and left incubated overnight at 37°C.

Figure 4: Growth (swarm) diameter of swarm assay in Figure 2.