Composite

Part:BBa_K3710005

Designed by: Sandra Lizeth Martinez Diaz, Erik Hanko and Katherine Baker   Group: iGEM21_Manchester   (2021-10-01)
Revision as of 08:17, 21 October 2021 by LRoche (Talk | contribs)


Sarcosine Biosensor

This part encodes the biosensor genetic circuit comprising the sarcosine-specific transcription factor SouR and the promoter region pglyA1.

The part was meant to serve as the starting biosensor circuit platform for the subsequent creatinine biosensors that can be used in living therapeutics.

Pseudomonas aeruginosa encodes a sarcosine oxidase catabolic operon (sox) comprising the sarcosine oxidase genes involved in the creatinine degradation pathway. Sarcosine (N-methylglycine) is generated from a number of catabolic pathways including the creatinine metabolism and serves as both a carbon and nitrogen source for growth. During pathogenesis and within its environment P. aeruginosa is able to metabolize sarcosine precursors including the herbicide glyphosate and creatine into sarcosine.

The Sarcosine Oxidation and Utilization Regulator (SouR) was first identified by Willsey et al (2016) using a transposon-based genetic screen of the sox operon in Pseudomonas aeruginosa and through β-galactosidase assays . SouR is the first bacterial transcriptional regulator showing a selective and tight induction in response to sarcosine or structurally related compounds (e.g. ethylglycine ). SouR is a member of the glutamine amidotransferase I-like transcription regulator (GATR) subfamily of the AraC regulator family (CD03137) and is encoded by the PA4184 gene of P. aeruginosa. Willsey et al (2016) demonstrated that SouR is essential for growth in sarcosine as an energy source and binds within the -210 and -158 bp upstream region from the glya1 translational start site. Although little is known about SouR and sox genes in gram negative bacteria besides their widespread distribution, it is likely that creatinine or creatine could also act as an inducing ligand of SoxR.

PromoterCharacterisation

We followed the protocols we made for fluorescence assays of our promoters found here: https://2021.igem.org/Team:Manchester/Wet-lab. The results obtained for Sarcosine can be seen below:

[[File:BBa_K3710005_Results.png|200px|thumb|left|alt text]] [[File:BBa_K3710005_Results.png]]
Figure 3 (Left): Bacterial growth at different sarcosine concentrations measured at time 0, 3, 6 and 9 hours. (Right): Normalised Fluorescence observed at varying Sarcosine concentrations.

The results observed for the sarcosine sensor are good and provide insight into the concentration of sarcosine which will be required for teams in the future to use this sarcosine inducible system..


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 87
    Illegal AgeI site found at 773
    Illegal AgeI site found at 885
    Illegal AgeI site found at 2785
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 3
    Illegal SapI site found at 190


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Categories
Parameters
None