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, 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). For this experiment, we included two biological replicates to test our experimental strain (GFP+RFP A and GFP+RFP B).

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 strains 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.

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 4CL is placed under a constitutive promoter.

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). For this experiment, we included two biological replicates to test our experimental strain (deNovo A & deNovo B). IPTG was used to induce production of the enzymes.

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).

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, 3, 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). IPTG was used to induce production of the enzymes.

Results
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.

Cells were grown in 7 mL LB (and relevant antibiotics) in a 50 mL Falcon tube at 25°C in the shaking incubator at 220 rpm. 100 µL of each sample was extracted at 0, 2, 4, 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). IPTG was used to induce production of the enzymes.

Results
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.

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.