Part:BBa_K581005

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Following is a contribution to this part, by the NUS_Singapore 2019 team.

Group

NUS_Singapore 2019

Author

Sheena Chan

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 (doi:10.1073/pnas.0708102104). This inspired team NUS 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. coli MG1655 growth, concentrations ranging from x to y 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 1h and read for 12h continuously using a microplate reader.