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OVERVIEW

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LIST OF COMPOSITE PARTS

Device	Part Number	Usage
PhtpG1-mRFP	BBa_K2819118	Stress reporter
NANDA FILL ME	BBa_I20270	Promoter MeasKit (J23151)

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PhtpG1-mRFP

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.

In our experiments, we were interested in the depletion of finite cellular resources during the expression of synthetic constructs constitutes an unwanted burden, which we define as cell stress, 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, a remarkable discovery which we believe will have significant implications in the biomanufacturing field.

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.

Characterization using using E. coli DH5α as the host

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, 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.

Characterization using using E. coli DH5α as the host

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, 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.

CLOSE FLUORESCENCE STANDARD CURVE (CALIBRATION 3)

Materials

• Fluorescein
• 10 ml 1X PBS pH 7.4 - 7.6
• 96-well plate (black)

Methods

(A) To prepare the fluorescein stock solution

1. The fluorescein kit tube was spun down to make sure that the pellet was collected at the bottom of the tube.
2. 10X fluorescein stock solution (100 µM) was prepared by resuspending fluorescein in 1 ml of 1X PBS. Fluorescein was checked to be properly dissolved in PBS by checking for no more visible particulates in the pipette tip when resuspending.
3. 10X fluorescein stock solution was diluted with 1X PBS to make a 1X fluorescein solution (10 µM): 100 µl of 10X fluorescein stock solution was mixed with 900 µl of 1X PBS.

Figure 3: Graph of fluorescence against fluorescein concentration.

(B) To prepare the serial dilution of fluorescein

1. 100 µl of PBS was added into wells A2, B2, C2, D2...A12, B12, C12, D12.
2. 200 µl of 1X fluorescein stock solution was added into A1, B1, C1 and D1.
3. 100 µl of 1X fluorescein stock solution was transferred from A1 to A2.
4. Mix A2 by pipetting up and down 3 times and transfer 100 µl into A3.
5. The subsequent dilutions were prepared as illustrated by Fig. 4.
6. Fluorescence of all samples were measured, and our results are reflected by Fig. 5.

Figure 4: Graph of fluorescence against fluorescein concentration, logarithmic scale.

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