Part:BBa_K2924018

Promoter fliC with the reporter gene eYFP

Promoter FliC (BBa_K2924016) expressing the reporter gene eYFP(BBa_E0030)

Usage and Biology

The iGEM team from Düsseldorf 2019 used the published promoter fliC ¹ and combined it with the reporter gene eYFP (BBa_E0030) to create an acyl-CoA sensitive in vivo biosensor. Acyl-CoA is the activated form of fatty acids. If fatty acids are present the blocked promoter fliC is activated and the reporter gene eYFP can be synthesized. The promoter fliC is to find in the native Escherichia coli genome and eYFP is a green/yellow fluorescent protein from the cnidaria Aequorea victoria which has an excitation maximum hat 513 nm and an emission maximum 527 nm. Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Fig.1: Promoter fliC with reporter gene eYFP in a pBb backbone. The backbone has a kanamycin resistance and a medium copy ori p15A. EcoRI and XbaI were used as restriction enzymes.

The promoter was cloned prior to the reporter gene into a pBbAk6 which contains an kanamycin antibiotic resistance cassette². The origin of replication is a p15A and thus the plasmid represents medium copy number. This part was cloned with the BioBrick restriction enzymes in frame to apply a universal cloning strategy and make it applicable for further iGEM teams. >

The promoter was tested for the sensitivity to butyric acid in the culture medium by combining the promoter to an eYFP (BBa_E0030)² as an reporter gene. The concentrations of butyric acid were from 0.5 mM to 20 mM.

Fig.2: Response of P_fliC+eYFP (red) to different chain lengths of fatty acids compared to an empty vector control (black). The fluorescence was measured at an excitation wavelength from 497 nm and an emission wavelength from 540 nm.

The experiment indicates that the fluorescence does not increase with higher concentrations of butyric acid. Surprisingly the fluorescence from the empty vector control rises with higher concentrations while the P_fliC shows a falling tendency.

References

1: Toru Tobe,* Noriko Nakanishi, and Nakaba Sugimoto “Activation of Motility by Sensing Short-Chain Fatty Acids via Two Steps in a Flagellar Gene Regulatory Cascade in Enterohemorrhagic Escherichia coli” INFECTION AND IMMUNITY, Mar. 2011, p. 1016–1024

2: Lee TS, Krupa RA, Zhang F, Hajimorad M, Holtz WJ, Prasad N, Lee SK, Keasling JD. ”BglBrick vectors and datasheets: A synthetic biology platform for gene expression.” J Biol Eng. 2011 Sep 20;5:12. 10.1186/1754-1611-5-12 PubMed 21933410