OVERVIEW

LIST OF COMPOSITE PARTS

PhtpG1-mRFP

Characterization using using E. coli DH5α as the host

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.