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.
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).
Characterization using E. coli BL21 (DE3) as the host
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 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.
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