Composite

Part:BBa_K2924017

Designed by: Mirko Kraus   Group: iGEM19_Duesseldorf   (2019-10-16)


Promoter aldA with the reporter gene eYFP

Promoter aldA (BBa_K2924015) expressing the reporter gene eYFP(BBa_E0030)

Usage and Biology

The iGEM team from Düsseldorf 2019 used the already published promoter aldA and combined it with the reporter gene eYFP (BBa_E0030) to create an acyl-CoA sensitive in vivo biosensor1. Acyl-CoA is the activated form of fatty acids. Once fatty acids are present the blocked promoter aldA is activated and the reporter gene eYFP can transcribed and translated onto a fluorescently dectable protein eYFP. The promoter aldA originates from the wild type Escherichia coli genome and eYFP is a yellow fluorescent protein from the cnidaria Aequorea victoria, which have an excitation maximum at 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 aldA 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.
Fig. 2: Response of paldA+eYFP (yellow) to different chain lengths of fatty acids compared to an empty vector control (gray). Plot A presents the response for palmitic acid, plot B for stearic acid and plot C for oleic acid. The fluorescence was measured at an excitation wavelength from 497 nm and an emission wavelength from 540 nm.

After adding oleic acid (C18:1) many genes showed an altered expression level and new proteins like AldA were synthesised. The promoter for aldA was also used for the production of green fluorescent protein (GFP) as a reporter 1. PaldA was isolated from the Escherichia coli wild type genome.





The sensitivity of the promoter to fatty acids was tested with the reporter gene eYFP (Part:BBa_E0030). Different long-chain fatty acids, like palmitic acid, stearic acid and oleic acid were used for the characterization. Also different concentrations from 0.01 mM to 1 mM were used of the fatty acid palmitic acid and stearic acid. For the fatty acid oleic acid, the concentrations from 0.5 mM to 10 mM were used.
















References

1: Mee-Jung Han, Jeong Wook Lee, Sang Yup Lee, and Jong Shin Yoo. “Proteome-Level Responses of Escherichia coli to Long-Chain Fatty Acids and Use of Fatty Acid Inducible Promoter in Protein Production” Hindawi Publishing Corporation Journal of Biomedicine and Biotechnology Volume 2008, Article ID 735101,12 pages

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

[edit]
Categories
Parameters
None