Difference between revisions of "Team:NUS Singapore-A/shadow/Composite Part"
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| − | + | 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. | |
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| + | 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 OD<sub>600</sub> 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). | ||
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Revision as of 11:42, 15 October 2018
OVERVIEW
To write an overview.
To continue writing something else.
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).
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.
You can put text here if you wanna
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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You can put text here if you wanna
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