Part:BBa_K1463704
MotA and B0032 RBS under J23106 promoter
MotA and B0032 RBS under J23106 promoter.
Usage and Biology
Our MotA biobrick with B0032 RBS was ligated into the pSB1C3 submission vector and also the plasmid J61002 containing the strong J23100 promoter. After transformation colonies were only obtained with the pSB1C3 vector. A repeated ligation into the vector containing the strong J23100 promoter gave two colonies. However, sequencing showed that while motA clones in pSB1C3 had the correct sequence, the two inserts downstream of BBa_J23100 had mutations. One contained a mutation in the ribosome binding site, while the other had a 5 base deletion at the 5' end of the motA gene. This suggested that promoter J23100 was too strong and that high levels of motA expression might be toxic to the cells.
We inserted the BBa_B0032 RBS – motA biobrick into the BBa_J61002 vector containing a variety of different promoters from the parts distribution: BBa_J23106 (½ the strength of J23100) BBa_J23116 (¼ the strength of J23100) BBa_J23103 (very weak promoter) BBa_J23112 (weakest promoter we could find in the registry, barely any expression) (Strength measured with RFP: Part BBa_J23100)
These were checked by DNA sequencing and all found to have the expected sequence.
We then used swarm assays (semi-solid agar motility test) to investigate whether these plasmids would rescue swimming of a motA mutant. DS941 ΔmotA (with motA deleted) was transformed with pSB1C3 motA (no promoter), motA transcribed from the four different weaker promoters in BBa_J61002, and also motA with the strong J23100 promoter with the mutated ribosome binding site. The results of the swarm assays are shown in Figures 1 and 3. DS941 and MG1655-Z1 (another positive swimming control) swam to approximately the same distance. Three different isolates of DS941 ΔmotA did not swim at all, as expected for this mutant knocked out for the MotA motor protein. However, none of the plasmids containing motA restored swimming to the mutant to any significant extent, although it is possible that pSB1C3-motA (with no promoter) and BBa_J23100 – motA plasmid (with mutant RBS) gave slightly more mobility than no plasmid at all (Figures 1 and 3).
MotA is expressed from an operon containing two flagellar motor genes, motA and motB, and both of these genes are required for motor function, and hence swimming. Deletions in upstream genes in operons are often known to have “polar” effects, disrupting expression of downstream genes. Therefore our motA deletion might be severely reducing expression of motB. To test this, we made a motA-motB biobrick and check whether it restores swimming to our delta-motA mutant.
The motA motB J23100 promoter construct didn't give any colonies but ligations with other promoters did, suggesting that the J23100 promoter is too strong, and over expression of motility proteins could be toxic. DS941 ΔmotA was transformed with BBa_J23103 motA motB, BBa_23106 motA motB, BBa_J23112 motA motB and BBa_J23116 motA motB all in the plasmid vector BBa_J61002. Gene rescue was checked again by doing swarm assay (Figure 5). This time we saw a significantly better result than just with motA, supporting our hypothesis that the motA mutation disrupts expression of motB.
The diameter of migration on the swarm plates is shown in the histograms in figure 2, 4 and 6. The distance migrated when motA and motB were introduced into DS941 ΔmotA correlated well with the strength of the promoters driving expression of motA and motB. The two stronger promoters BBa_J23116 and BBa_J23106 restored swimming to a greater extent than the two weaker promoters BBa_J23103 and BBa_J23112.
Fig. 2: Swarm assay. 5µ drop of overnight culture was added on a soft-agar plate and left incubated overnight at 37°C.
Fig. 3: DS941 ΔmotA E. coli carrying plasmids with the indicated biobricks were tested for mobility on swarm plates. Growth diameter of swarm assay grown for 16 hours at 37 degrees C. Non knock-out strains used as a controls.
Fig. 4: Swarm assay. 5µ drop of overnight culture was added on a soft-agar plate and left incubated overnight at 37°C. Different strength promoters used this time.
Fig. 5: DS941 ΔmotA E. coli carrying plasmids with the indicated biobricks were tested for mobility on swarm plates. Growth diameter of swarm assay grown for 16 hours at 37 degrees C. Non knock-out strain used as a control.
Fig. 6: Swarm assay. 5µ drop of overnight culture was added on a soft-agar plate and left incubated overnight at 37°C. Both motA and motB under different strength promoters.
Fig. 7: DS941 ΔmotA E. coli carrying plasmids with the indicated biobricks were tested for mobility on swarm plates. Growth diameter of swarm assay grown for 16 hours at 37 degrees C. Non knock-out strain used as a control.
More information on the biobrick and usage:
http://2014.igem.org/wiki/index.php?title=Team:Glasgow/Project/Mobility_Proteins#motA
https://parts.igem.org/Part:BBa_K1463700
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 385
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 123
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 891
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