Difference between revisions of "Part:BBa K581005"

Revision as of 09:10, 4 October 2019

Pc+sgrS(wt)+Terminator (small RNA regulator, conjugate part of ptsG(wt))

SgrS(sugar transport-related sRNA)(wt) is a small RNA regulator that help cells recover from glucose-phosphate stress by base pairing with ptsG(wt)[1] mRNA. SgrS regulates ptsG mRNA by short, imperfect base-pairing interactions; as a result, the expression of PtsG is repressed.

Teppei Morita et.al’ s work suggests that two mutations (C85G and C87G) in ptsG mRNA could completely impair the ability of SgrS to downregulate its expression, while compensatory mutations of SgrS (G178C and G176C) restore the gene silencing ability. These results indicate that it is the base pairing of the two RNAs rather than particular nucleotides that is important for SgrS action. They have also illustrated that sequence outside this region, even though complementary, is rather dispensable for the efficient silencing (Kawamoto et al., 2006). This makes mutant ptsG/SgrS pairs orthogonal to genetic context of the host cell. Therefore we choose this couple of conjugate mRNA/sRNA as the foundation of our comparator device design.

SgrS(wt) in this part will be constitutively expressed in E.coli after transformation processing.

                 
                Fig.1 Sequence alignment of wildtype ptsG/SgrS pair and its mutant complementary pairs. 

Sequence and Features

Group

NUS_Singapore 2019

Summary and Uploads

Team NUS 2019 has contributed additional characterization data for biobrick BBa_K581005 which is supposed to repress the expression of ptsG in cell. In 2007, Wadler and Vanderpool reported SgrS’ ability to inhibit growth in E. coli, especially when glucose is the only carbon source (Wadler & Vanderpool, 2007). This inspired team NUS Singapore 2019 to explore SgrS as a potential candidate to fine tune the growth rate of E. coli MG1655 so as to prolong the overall viable lifespan of E. coli.

Looking at past characterization data of this biobrick which utilized a constitutive promoter to express SgrS, team NUS 2019 adopted tetracycline inducible system to control the expression of SgrS instead. As the team was interested to study the effect of varying anhydrotetracycline (aTc) concentrations on E. coliMG1655 growth and protein production, we co-transformed RFP regulated by IPTG inducible system to validate protein production. Concentrations of aTc ranged from 0 to 1.5mM were investigated. The cells were grown in LB media at 37°C, washed with M9 media (0% glucose) and finally characterized in M9 with 0.2% glucose at a starting OD₆₀₀ of 0.1 at 0h. Cells were induced with respective aTc concentrations at 0h and 150uM of IPTG at 1hr, which was then read for 20h continuously using a microplate reader to compare growth (Figure 1) and total RFP produced (Figure 2). Furthermore, we normalized RFP to OD₆₀₀ to determine the maximum RFP production per cell (Figure 3).



Figure 1: Growth curve of MG1655 SgrS_A6A induced with aTc (0nm, 10nm, 100nm and 1500nm) at 0h and 150uM of IPTG at 1h



Figure 2: Total RFP of MG1655 SgrS_A6A induced with aTc (0nm, 10nm, 100nm and 1500nm) at 0h and 150uM of IPTG at 1h



Figure 3: RFP per OD₆₀₀ of MG1655 SgrS_A6A induced with aTc (0nm, 10nm, 100nm and 1500nm) at 0h and 150uM of IPTG at 1h

With these results, the effects of SgrS on cells affects its growth which directly influences the protein production; with higher concentrations of aTc producing lesser intensity of RFP and vice versa.

Reference: Wadler, C. S., & Vanderpool, C. K. (2007). A dual function for a bacterial small RNA : SgrS performs base pairing-dependent regulation and encodes a functional polypeptide. PNAS, 104(51), 20454–20459.

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