Composite

Part:BBa_K1463702

Designed by: Beth Greig   Group: iGEM14_Glasgow   (2014-10-04)

MotA with B0032 RBS under J23100 promoter

MotA with B0032 RBS under J23100 promoter.


Usage and Biology

Our MotA biobrick K1463700 with B0032 RBS was ligated into the plasmid J61002 containing the strong J23100 promoter. After first transformation colonies were not obtained. A repeated ligation into the vector containing the strong J23100 promoter gave two colonies. However, sequencing showed that 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 then used swarm assays (semi-solid agar motility test) to investigate whether this plasmid would rescue swimming of a motA mutant. DS941 ΔmotA (with motA deleted) was transformed with pSB1C3 motA (no promoter) and motA with the strong J23100 promoter with the mutated ribosome binding site. The results of the swarm assays are shown in Figures 1. 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. 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), but they have not restored swimming completely.

See BBa_K1463700 for more details.

288px-GU_Gintare_Plates_1_Small.png

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

GU_Gintare_illutration_4.png

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.



For more information on the biobrick and usage go to:

http://2014.igem.org/wiki/index.php?title=Team:Glasgow/Project/Mobility_Proteins#motA

http://parts.igem.org/Part:BBa_K1463700

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 385
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 123
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 891


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