Part:BBa_K581005:Experience

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

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/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.