Difference between revisions of "Part:BBa K2819110"

Latest revision as of 06:06, 14 October 2018

PhtpG1 Burden-Driven Promoter

The promoter, PhtpG1, was carefully chosen because of sensitivity to synthetic construct-induced burden in E. coli; the depletion of finite cellular resources during the expression of synthetic constructs constitutes an unwanted burden, hampering the growth and expected the performance of engineered cells in an unpredictable manner. This distinct characteristic is especially valuable to our system because we were interested in quantifying real-time levels of stress generated by the expression of externally introduced constructs. By quantifying cell stress via fluorescence, recombinant protein production can be optimized by the user simply by reducing cell stress i.e. switching off protein production (in our case, this can be done by turning on blue light). Additionally, according to Ceroni et al. (2018), PhtpG1 displayed the best on/off characteristic out of the 4 promoters that were being investigated (htpG1, htpG2, groSL, and ibpAB). This feature allows the promoter to not only respond rapidly but also to maintain its receptivity in a dynamic cell microenvironment.

This promoter has been characterized in depth by attaching the mRFP gene downstream the promoter to express red fluorescence protein (RFP) in response to its level of activation. Please refer to page: https://parts.igem.org/Part:BBa_K2819118 to read more about how this promoter can be useful.

For more information about the burden-driven feedback mechanism, please visit http://2018.igem.org/Team:NUS_Singapore-A.

Sequence and Features

Sequence and Features

Usage and Biology

• Stress externally introduced via a synthetic construct can be quantified via this part. Levels of fluorescence will be dependent on the amount of stress experienced by the cell since transcription will be driven by the stress-induced promoter.
• Basal stress levels (inherent stress levels, in addition to the stress brought about by the PhtpG1-mRFP construct itself) can be measured.
• Characterization was done in DH5α and BL21 Star (DE3). Ceroni et al. (2018) have also successfully tested the PhtpG1 promoter in MG1655 and DH10B.
• To note: Plasmid backbone part that is inserted should not have the same antibiotic resistance as the bacteria it is transformed into.

Characterization

Summary of Results as in https://parts.igem.org/Part:BBa_K2819118

• PhtpG1-mRFP can work in different genetic backgrounds. Interestingly, the stress reporter was robust in all three strains of E. coli that it was tested in: DH5α, BL21 (DE3) and BL21 Star (DE3).
• PhtpG1-mRFP is robust in different temperatures. However, at low temperatures i.e. 25°C, more time is expected to allow for growth to stabilize/increase steadily before readings follow an expected trend. Given that PhtpG1 is a promoter that is involved in heat-shock response (and that requires heat-shock response sigma factor (σ32)), higher temperatures would be expected to activate the promoter to a greater extent (Ceroni et al., 2018).
• PhtpG1-mRFP appears to be sensitive to the size of constructs that is introduced into the cell. The highest levels of mRFP was consistently found to be generated in test construct Set-up C (see Figure 6), in which the large de novo plasmid^a was expressed, followed by test construct Set-up E and F expressing the moderately sized FNS^a protein, and the lowest being in test construct Set-up A, in which only GFP was expressed^a.

^aSizes of proteins: MCS (55 kDa) [de novo plasmid], OsPKS (43 kDa) [de novo plasmid], 4CL (59 kDa) [de novo plasmid], GFP (27 kDa) and FNS (41 kDa).

References

Ceroni, F., Boo, A., Furini, S., Gorochowski, T.E., Borkowski, O., Ladak, Y.N., Awan, A.R., Gilbert, C., Stan, G.B., and Ellis, T. (2018). Burden-driven feedback control of gene expression. Nat. Methods. Published March 26, 2018. https://doi.org/10.1038/nmeth.4635.

Leonard, E., Yan, Y., Fowler, Z. L., Li, Z., Lim, C.-G., Lim, K.-H., & Koffas, M. A. G. (2008). Strain Improvement of Recombinant Escherichia coli for Efficient Production of Plant Flavonoids. Mol. Pharmaceutics, 5(2), 257–265. http://doi.org/http://dx.doi.org/10.1021/mp7001472