Difference between revisions of "Part:BBa K3710005"

Revision as of 20:15, 21 October 2021

Sarcosine Biosensor

This part harbours the sarcosine biosensor composed of the sarcosine-responsive transcriptional regulator SouR and the sarcosine-inducible promoter PglyA1.

Pseudomonas aeruginosa encodes a sarcosine oxidase catabolic operon (sox) comprising the sarcosine oxidase genes involved in the creatinine degradation pathway [1]. 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 metabolise 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) [1] using a transposon-based genetic screen of the sox operon in Pseudomonas aeruginosa and confirmed by β-galactosidase reporter 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 on sarcosine as an energy source and binds within the -210 and -158 bp upstream region from the glyA1 translational start site [1]. 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:

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..

References:

[1] Willsey, G. G., & Wargo, M. J. (2016). Sarcosine catabolism in Pseudomonas aeruginosa is transcriptionally regulated by SouR. Journal of Bacteriology, 198(2), 301-310.

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

@@ Line 20: / Line 20: @@
 <p>This part harbours the sarcosine biosensor composed of the sarcosine-responsive transcriptional regulator SouR and the sarcosine-inducible promoter PglyA1.</p>
-<p>The part was meant to serve as the starting biosensor circuit platform for the subsequent creatinine biosensors that can be used in living therapeutics.</p>
+<p>Pseudomonas aeruginosa encodes a sarcosine oxidase catabolic operon (sox) comprising the sarcosine oxidase genes involved in the creatinine degradation pathway [1]. 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 metabolise sarcosine precursors including the herbicide glyphosate and creatine into sarcosine.</p>
-<p>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 metabolise sarcosine precursors including the herbicide glyphosate and creatine into sarcosine.  </p>
+<p>The Sarcosine Oxidation and Utilization Regulator (SouR) was first identified by Willsey et al. (2016) [1] using a transposon-based genetic screen of the sox operon in Pseudomonas aeruginosa and confirmed by β-galactosidase reporter 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 on sarcosine as an energy source and binds within the -210 and -158 bp upstream region from the glyA1 translational start site [1]. 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.</p>
-<p>The Sarcosine Oxidation and Utilization Regulator (SouR) was first identified by Willsey et al. (2016)[1] using a transposon-based genetic screen of the sox operon in Pseudomonas aeruginosa and through  &#946;-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 on sarcosine as an energy source and binds within the -210 and -158 bp upstream region from the glyA1 translational start site [1]. 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.</p>
 <h2>PromoterCharacterisation</h2>
@@ Line 43: / Line 41: @@
 References:
-Willsey, G. G., & Wargo, M. J. (2016). Sarcosine catabolism in Pseudomonas aeruginosa is transcriptionally regulated by SouR. Journal of Bacteriology, 198(2), 301-310.
+[1] Willsey, G. G., & Wargo, M. J. (2016). Sarcosine catabolism in Pseudomonas aeruginosa is transcriptionally regulated by SouR. Journal of Bacteriology, 198(2), 301-310.
 <!-- Add more about the biology of this part here