Part:BBa_K2819118

Stress Reporter: mRFP Gene under Burden-Driven Promoter

This part contains the coding sequence of mRFP put under control of the stress promoter PhtpG1. The promoter, PhtpG1, was carefully chosen because of sensitivity to synthetic construct-induced burden in E. coli. 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 stress-reporting module, PhtpG1-mRFP, to not only respond rapidly, but also to maintain its receptivity in a dynamic cell microenvironment.

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.

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

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 662
  Illegal AgeI site found at 774
  
• 1000
  COMPATIBLE WITH RFC[1000]

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 6: 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.

Figure 1: PCDF-RFP (red) is a control strain as in Setup B in Figure 6 (see below) that carries only the stress promoter module. GFP+RFP (blue/yellow) carries both stress promoter module and Pcon-GFP as in Set-up A in Figure 6 (see below). (A) mRFP values measured in fluorescence (RFU) per OD600 (a.u.) over a period of 24 hours. (B) mRFP(FU)/OD(a.u.) values at 24 h time point. (C) GFP values measured in fluorescence (GFU) per OD600 (a.u.) over a period of 24 hours. (D) GFP (FU)/OD (a.u.) values at 24 h time point.

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.

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
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, 7 h time points in triplicates to measure fluorescence (GFP/mRFP) and OD₆₀₀ using microplate reader (BioTek). All values were corrected by using LB and respective antibiotics as blanks (streptomycin and/or kanamycin and/or ampicillin).

Results
We were able to show that the de novo construct which expresses three enzymes OsPKS MCS and 4CL as compared to just GFP activated the stress promoter, PhtpG1, to a greater extent. This is manifested in higher levels of mRFP measured over a time period of 24 hours (see Figure 2A) 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 B (see Figure 2C). From this data, we were able to deduce that larger externally induced construct which expresses larger or more foreign proteins cause greater cell stress than smaller constructs carrying genes of smaller proteins (i.e., GFP).

Figure 2: PCDF-RFP (red) is a control strain as in Setup B in Figure 6 (see below) that carries only the stress promoter module. RFP+GFP (blue) carries both stress promoter module and Pcon-GFP as in Set-up A in Figure 6 (see below). deNovo A & B (yellow/green) carry the naringenin de novo plasmid as in Set-up C in Figure 6 (see below). (A) mRFP values measured in fluorescence (FU) per OD₆₀₀ (a.u.) over a period of 24 hours. (B) RFP(FU)/OD(a.u.) values at 24 h time point. (C) GFP values measured in fluorescence (FU) per OD₆₀₀ (a.u.) over a period of 24 hours. (D) GFP(FU)/OD(a.u.) values at 24 h time point.

Characterization using E. coli BL21 (DE3) as the host at 25°C vs. at 37°C
We were also interested in characterizing our stress reporting module against different genetic backgrounds. In this set of experiments, BL21 (DE3) was used. 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
Cells were grown in 7 mL LB (and relevant antibiotics) in a 50 mL Falcon tube at 37°C and 25°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 in triplicates to measure fluorescence (GFP/mRFP) and OD₆₀₀ using microplate reader (BioTek). All values were corrected by using LB and respective antibiotics as blanks (streptomycin and/or kanamycin and/or ampicillin).

Results at 37°C
Changing the host strain from DH5α to BL21 (DE3) did not change the trend observed that larger constructs i.e. de novo plasmid led to greater mRFP production. The highest mRFP levels were observed in deNovo (see Figure 3A) over the entire 24-hour period. We also recorded GFU per OD₆₀₀ to confirm that GFP was only expressed in GFP+RFP and not in deNovo (see Figure 3D). From this data, we were able to deduce that our stress reporter was robust in different genetic backgrounds.

Figure 3: PCDF-RFP (red) is a control strain as in Setup B in Figure 6 (see below) that carries only the stress promoter module. GFP+RFP (blue) carries both stress promoter module and Pcon-GFP as in Set-up A in Figure 6 (see below). deNovo (yellow) carry the naringenin de novo plasmid as in Set-up C in Figure 6 (see below). (A) mRFP values measured in fluorescence (FU) per OD600 (a.u.) over a period of 24 hours. (B) RFP(FU)/OD(a.u.) values at 24 h time point. (C) GFP values measured in fluorescence (FU) per OD₆₀₀ (a.u.) over a period of 24 hours. (D) GFP(FU)/OD(a.u.) values at 24 h time point.

Results at 25°C
Changing the temperature at which the experiment was conducted from 37°C to 25°C also did not change the trend observed at the 24 hour time point. The expression of the de novo plasmid generated the highest level of mRFP per OD₆₀₀, followed by the expression of Pcon-GFP (see Figure 4B). Albeit sharing the same trend, the absolute mRFP per OD₆₀₀ values are lower than the values observed in the experiment conducted at 37°C.

Additionally, the overall trend of mRFP levels per OD₆₀₀ over the 24 hour time period differed slightly from that of being observed in the experiment conducted at 37°C. 25°C, being a lower temperature, evidently slowed down growth in the cells, which in turn, explains why in hours 2-6, mRFP/GFP per OD₆₀₀ were unusually high since fluorescence levels were divided by very small OD₆₀₀ values. After the 6 hour mark, there is a steady increase in mRFP per OD₆₀₀ levels for all three samples, following which, the trend returns as is expected. From this data, we were able to deduce that our stress reporter was robust in different temperatures.

Figure 4: PCDF-RFP (red) is a control strain as in Setup B in Figure 6 (see below) that carries only the stress promoter module. GFP+RFP (blue) carries both stress promoter module and Pcon-GFP as in Set-up A in Figure 6 (see below). deNovo (yellow) carry the naringenin de novo plasmid as in Set-up C in Figure 6 (see below). (A) mRFP values measured in fluorescence (FU) per OD600 (a.u.) over a period of 24 hours. (B) RFP(FU)/OD(a.u.) values at 24 h time point. (C) GFP values measured in fluorescence (FU) per OD600 (a.u.) over a period of 24 hours. (D) GFP(FU)/OD(a.u.) values at 24 h time point.

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 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
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 in triplicates 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 and/or ampicillin and/or chloramphenicol).

Results
The expression of FNS generates a comparatively greater level of mRFP expression compared to the production of just GFP, but a lower level of mRFP expression compared to the large de novo plasmid (see Figure 4). This is expected given that the de novo plasmid carries three genes coding for OsPKS MCS and 4CL while Brep-FNS and pBAD-FNS only carries EL222/FNS and FNS respectively. The difference in mRFP per OD600 levels between Brep-FNS as compared to pBAD-FNS can be explained by the additional protein, EL222, expressed only in the Brep-FNS test construct (see Figure 6, Set-up E). This further supports our hypothesis that PhtpG1-mRFP is sensitive to the size/amount of of foreign proteins expressed in the cell.

Figure 5: PCDF-RFP (red) is a control strain as in Setup B in Figure 6 (see below) that carries only the stress promoter module. GFP+RFP (blue) carries both stress promoter module and Pcon-GFP as in Set-up A in Figure 6 (see below). deNovo (yellow) carry the naringenin de novo plasmid as in Set-up C in Figure 6 (see below). Brep-FNS (green) and pBAD-FNS are test constructs Set-up E and Set-up F respectively as in Figure 6, carrying both stress-promoter and Brep-FNS and pBAD-FNS respectively. (A) mRFP values measured in fluorescence (FU) per OD600 (a.u.) over a period of 24 hours. (B) RFP(FU)/OD(a.u.) values at 24 h time point. (C) GFP values measured in fluorescence (FU) per OD600 (a.u.) over a period of 24 hours. (D) GFP(FU)/OD(a.u.) values at 24 h time point.

Experimental Set-Ups

Figure 6. Experimental set-up to test for stress caused by the expression of externally introduced synthetic constructs. (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

• 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<b><u> 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 plasmida is expressed, followed by test construct Set-up E and F expressing the moderately sized FNSa protein, and the lowest being in test construct Set-up A, which expresses only GFP^a.</li> </ul>
  
  ^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).
  
  <b>Future Work</b>
  • It would be interesting

  
  <b>References</b>
  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

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