Composite

Part:BBa_K1463604:Design

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

FliC with RBS and J23100 promoter


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 BamHI site found at 1306
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 383
    Illegal AgeI site found at 791
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes


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.


Oligonucleotide Sequence

J23100_oligos.png
PINK: Prefix
COLOURLESS: J23100 promoter
GREY/BLUE: Scar sequence
RED: B0032 RBS

Flic Motility Swarm Assay

310px-GU_Figure_1_swarm_M.png

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)


GU_Figure_2_Motility_histogram.png
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


Source

E. coli DS941 (AB1157 derivative) genomic DNA