Part:BBa_K1463602:Design

FliC with RBS and J23116 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 J23116 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 EcoR1 and Xba1. This results in the incorporation of the J23116 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.

Oligonucleotide Sequence

PINK: Prefix
COLOURLESS: J23116 promoter
GREY/BLUE: Scar sequence
RED: B0032 RBS

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

Source

E. coli DS941 (AB1157 derivative) genomic DNA