Team:NUS Singapore-A/shadow/Composite Part
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) |
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
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.
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 E. coli BL21 (DE3) as the host
Abs600 |
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Fluorescence |
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Characterization using E. coli BL21 Star (DE3) as the host
Abs600 |
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Fluorescence |
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CONCLUSION
The results from our experiment seem to indicate that normalizing fluorescence measurements to absolute cell count using the Study’s two methods will not be able to reduce lab-to-lab variability because counting colony-forming units do not return the expected cell concentration, i.e. the cell concentration modeled by the silica beads in Method 1. While both methods cannot be used independently to establish a robust fluorescence measurement system, it may be possible that lab-to-lab variability can be reduced if a different method of normalizing to absolute cell count is devised, replacing Method 1, Method 2, or both.
HEAR OUR STORY
We’re using synthetic biology to change the way the world thinks and functions.
We’re a little disruptive, dangerous and maybe a tad too bold. But the world needs some shaking up every now and then, and we think we’re just the right people for that.
Together we’re going to engineer biology and create something awesome.
CONTACT US
nusigem@gmail.com21 Lower Kent Ridge Rd, Singapore 119077