FliC with RBS and J23100 promoter
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 7
Illegal NheI site found at 30
- 21Illegal BamHI site found at 1306
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 383
Illegal AgeI site found at 791
- 1000COMPATIBLE WITH RFC
This composite part was made by inserting a synthesised double-stranded oligonucleotide containing J23100 promoter and B0032 RBS into K1463601 (fliC and B0034 RBS). The oligonucleotide sequence is shown below. After annealing top and bottom strands, the design of these oligos incorporated overhangs which corresponded to EcoRI and XbaI cut sites, allowing the oligonucleotide to be ligated into K1463601 cut with EcoRI and XbaI. This results in the incorporation of the J23100 promoter, the B0032 RBS and appropriate prefix/suffix/scar sites. The incorporation of two RBS was due to an oversight. However, as shown in Figures 1 and 2, this did not inhibit the restoration of swimming in the fliC knockout strain.
However, this particular promoter construct was found to be unable to restore swimming in the knockout strain. When later sequenced, this plasmid was found to have a mutation within the promoter, explaining this result. We failed to clone a functional J23100 promoter in front of the fliC biobrick, suggesting that strong over expression of fliC may be toxic.
Flic Motility Swarm Assay
Figure 1: FliC Swarm Motility Assays.
(A) DS941, (B) DS941 ΔfliC,
(C) DS941 ΔfliC + pSB1C3 fliC (no promoter), (D) DS941 ΔfliC + J23100 (mutant promoter) fliC,
(E) DS941 ΔfliC + J23116-fliC(1), (F) DS941 ΔfliC + J23116-fliC(2),
(G) DS941 ΔfliC + J23106-fliC(1), (H) DS941 ΔfliC + J23106-fliC(2)
Figure 2 - FliC Motility Histogram
For more information on the biobrick and methods used go to http://2014.igem.org/wiki/index.php?title=Team:Glasgow/Project/Mobility_Proteins#fliC
E. coli DS941 (AB1157 derivative) genomic DNA