Difference between revisions of "Part:BBa K2819118"

Revision as of 18:52, 10 October 2018

Stress Reporter: mRFP Gene under Burden-Driven Promoter

This part contains the coding sequence of mRFP put under control of the stress-inducible promoter PhtpG1. According to Ceroni et al. (2018), PhtpG1 is considered to be an “intrinsic biosensor for synthetic construct-induced burden in E. coli” which displayed the best on/off characteristic out of the 4 promoters that were being investigated (htpG1, htpG2, groSL, and ibpAB promoters). It was also proven to be highly sensitive and responsive to synthetic construct expression of foreign proteins. This is manifested in a significant increase in cell stress measured.

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. Stress regulation has been shown to enable cells to outperform their unregulated counterparts in terms of protein yield.

This stress-reporting module is especially important to our system because of its usefulness in providing information about the real-time status of the cells in a culture. 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).

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

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

Characterization using E. coli DH5α as the host
Characterization using Pcon-GFP
To show that our stress reporter part is sensitive to externally introduced constructs which produce foreign proteins (i.e., GFP), we set up an experiment as described in the methods below. Figure ###: A, B (below) shows the different test constructs that were used in the experiment. We were interested in stress induced by GFP production, in particular, because of its universal use as a reporter. Through this set of experiment, we aimed to find out if GFP production indeed leads to increase levels in cell stress.

Methods
Cells were grown in 7 mL LB (and relevant antibiotics) in a 50 mL Falcon tube at 37°C in the shaking incubator at 220 rpm. 100 µL of each sample was extracted at 0, 2, 4, 5, 6, 24 h time points to measure fluorescence (GFP/RFP) and OD₆₀₀ using microplate reader (BioTek). All values were corrected by using LB and respective antibiotics as blanks (streptomycin and/or kanamycin).

Results
Figure 1A shows that there is an overall trend of increased RFU per OD₆₀₀ over time. This is indicative of increased cell stress over time since transcription of the mRFP gene is under the stress-inducible promoter, PhtpG1. By comparing fluorescence units per OD₆₀₀ between control and experimental strain at the 24 h time point (see Figure 1B), we demonstrated that GFP production in cells caused about a 0.5 fold increase in RFU per OD levels, suggesting that there is an equivalent increase in cell stress. This data shows that our stress-reporting module PhtpG1-mRFP is not only successful in reporting cell stress but also sensitive and responsive to the presence of externally introduced constructs.

In order to confirm that GFP production contributed to the increase in RFP levels in the cell, we had to prove that GFP was properly expressed. To do so, we measured GFP levels (FU) per OD₆₀₀. Figure 1C illustrates that GFU per OD₆₀₀ in the control strain remains consistently low with little additional increase. This data shows that the control strain does not produce any GFP as is expected. GFU per OD₆₀₀ in strains GFP + RFP A and GFP + RFP B increase over time, demonstrating that GFP production within these two strains were successful. This is more clearly presented in Figure 1D, in which GFU per OD₆₀₀ levels at the 24 hour time point for strains GFP+RFP A and GFP+RFP B are substantially higher than that of the control strain. This, when coupled with results in Figure 1A (elaborated in section above), help prove that GFP production caused an increase in RFP levels in cells.

Characterization using De Novo Plasmid
This set of experiments is an extension of ‘Characterization using Pcon-GFP’. Having shown that GFP production does cause an increase in RFP levels in cells, which is indicative of additional cell stress, we then wanted to determine if larger constructs (i.e., our de novo plasmid) would cause greater burden in the cell and a corresponding increase RFP production. We hypothesized that larger constructs that carry multiple genes encoding for larger proteins would cause greater RFP expression.

De Novo Plasmid
This plasmid was designed to produce naringenin from tyrosine, a process involving catalysis by 4 enzymes - PAL, 4CL, OsPKS and MCS - put together in a plasmid. At current, our de novo construct already carries 3 of the 4 enzymes necessary for naringenin production: OsPKS and MCS are strategically placed under a Plac promoter while PAL is placed under a constitutive promoter. Visit our website for more information about the use of this plasmid in the context of our system at http://2018.igem.org/Team:NUS_Singapore-A.

Methods
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Results
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Characterization using E. coli BL21 Star (DE3) as the host
We were also interested in characterizing our stress reporting module against different genetic backgrounds. In this set of experiments, BL21 Star (DE3) was used. Given that BL21 Star (DE3) is a RNase knockout strain of E. coli, enzymes can be transcribed with a longer half-life of their respective mRNA (Leonard et al., 2008).

Characterization using De Novo Plasmid
Methods
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Results
Similarly, we showed that the larger de novo construct activated the stress promoter, PhtpG1, to a greater extent than GFP, which manifested in higher levels of RFP measured over a time period of 24 hours (see Figure 3A) in the cell expressing the three de novo enzymes as compared to the cell expressing GFP only. We also recorded GFU per OD₆₀₀ to confirm that GFP was only expressed in RFP + GFP A and not deNovo A or B (see Figure 3C).

Interestingly, in BL21 Star (DE3), we observed that the expression of GFP had little effect on RFP levels (see Figure 3A) as compared to GFP production in DH5α. This is more clearly elucidated at the 24 time point as in Figure 3B (### ADD). This data shows that BL21 Star (DE3) has a higher tolerance to cell stress than DH5α.

Characterization using Pcon-GFP & PBrep-FNS/PBAD-FNS
Given that characterization of the stress reporting module in DH5α was successful i.e. we were able to demonstrate that externally introduced synthetic constructs led to greater production of RFP in cells (RFP levels being deemed to be indicative of cell stress), we set out to measure the level of stress generated by the introduction of two constructs involved in the production of apigenin.

Methods
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Results
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Characterization at 25°C vs. at 37°C
Additionally, we were interested in how robust the promoter is at different temperatures. By establishing that the promoter functions in different temperatures, users may choose to utilize this part in a range of different experiments that may require to be conducted at different temperatures.

Methods
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Results
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Figure ###. Experimental set-up to test for stress caused by GFP production in cells. (A) Cell carrying stress reporter part (PhtpG1-mRFP) and GFP gene under Pcon. (B) Control cell carrying only stress reporter to measure basal levels of stress in cells that do not carry additional externally introduced constructs. (C) Cell carrying stress reporter and de novo plasmid¹. (D) Cell carrying stress reporter, de novo plasmid and GFP gene. (E) Cell carrying stress reporter and PBrep-FNS². (F) Cell carrying stress reporter and PBAD-FNS³.

¹De novo plasmid, when induced, produces enzymes that enables naringenin production. For further details, visit http://2018.igem.org/Team:NUS_Singapore-A.
²PBrep-FNS and ³PBAD-FNS, when induced, produces enzymes that enable the production of apigenin. For further details, visit http://2018.igem.org/Team:NUS_Singapore-A.

Conclusion

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

Functional Parameters