Difference between revisions of "Part:BBa K3710005"
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| − | <p>This part | + | <p>This part harbours the sarcosine biosensor composed of the sarcosine-responsive transcriptional regulator SouR and the sarcosine-inducible promoter PglyA1.</p> |
| − | <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> | + | <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> | + | <h2>Sensor Characterisation</h2> |
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| + | <p>To determine the range of detection of the sarcosine-sensor, E. coli carrying the sensor were grown in minimal media and left untreated or supplemented with sarcosine at final concentrations ranging from 0.2 to 100 mM. Fluorescence and absorbance were quantified every 5 min for 16 hours. First, we evaluated whether supplementation of the growth media with sarcosine had any effects on cell viability. Lower concentrations of up to 25 mM of sarcosine had a growth-promoting effect on E. coli especially during later growth stages (Figure 1, left inlet). The promoter output in response to a range of different sarcosine concentrations was quantified for cells in the exponential growth phase and normalised by culture absorbance. The sarcosine sensor showed an increase in RFP fluorescence with increasing concentration of sarcosine (Figure 1, right inlet). In the absence of inducer, fluorescence was at the level of media autofluorescence, indicating a tight repression of the glyA1 promoter by SouR. The minimum concentration of sarcosine that mediated an activation of reporter was 25 mM.</p> | ||
<img src="https://2021.igem.org/wiki/images/6/61/T--Manchester--dspbfig1.png"> | <img src="https://2021.igem.org/wiki/images/6/61/T--Manchester--dspbfig1.png"> | ||
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| + | <figcaption>Figure 1: Bacterial growth at different sarcosine concentrations measured at time 0, 3, 6 and 9 hours (left). Normalised Fluorescence observed at sarcosine concentrations ranging from 0 to 100 mM (right).</figcaption> | ||
| + | <p>The SouR/PglyA1-sarcosine sensor mediated controllable gene expression upon supplementation with sarcosine above 25 mM. However, for the sensor to be used within our kill switch design, it would be required to respond to μM concentrations of sarcosine to mimic the conditions of the urinary tract. Protein engineering could be performed on SouR aiming at increasing its sensitivity for sarcosine.</p> | ||
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| + | References: | ||
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| + | [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 | <!-- Add more about the biology of this part here | ||
Latest revision as of 20:29, 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.
Sensor Characterisation
To determine the range of detection of the sarcosine-sensor, E. coli carrying the sensor were grown in minimal media and left untreated or supplemented with sarcosine at final concentrations ranging from 0.2 to 100 mM. Fluorescence and absorbance were quantified every 5 min for 16 hours. First, we evaluated whether supplementation of the growth media with sarcosine had any effects on cell viability. Lower concentrations of up to 25 mM of sarcosine had a growth-promoting effect on E. coli especially during later growth stages (Figure 1, left inlet). The promoter output in response to a range of different sarcosine concentrations was quantified for cells in the exponential growth phase and normalised by culture absorbance. The sarcosine sensor showed an increase in RFP fluorescence with increasing concentration of sarcosine (Figure 1, right inlet). In the absence of inducer, fluorescence was at the level of media autofluorescence, indicating a tight repression of the glyA1 promoter by SouR. The minimum concentration of sarcosine that mediated an activation of reporter was 25 mM.
The SouR/PglyA1-sarcosine sensor mediated controllable gene expression upon supplementation with sarcosine above 25 mM. However, for the sensor to be used within our kill switch design, it would be required to respond to μM concentrations of sarcosine to mimic the conditions of the urinary tract. Protein engineering could be performed on SouR aiming at increasing its sensitivity for sarcosine.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 3
Illegal SapI site found at 190