Team:NCTU Formosa/Proof Of Concept
Overview
Concept:
Gloeobacter rhodopsin with light induced contributes to additional ATP production in vivo which reduce the metabolic stress caused by excessive reliance on glucose and support bioengineering reactions. Therefore, E. Hybrid serves as a
fundamental strain use light as energy source for better growth and target protein expression.
How do we prove it?
We proved our concept with a meticulous process which can be roughly divided into three parts: Model, Lab Work, and Demonstration. Combined modelling results and predictions with our lab work and software, we enable to make E. Hybrid
work as we imagined. We could further prove that GR can be implemented to the real world for daily usages.
Model
More information in Model
We constructed logical ODEs to describe the overall growth curves and glucose consumption of wild type E. coli and E. Hybrid to prove the concept. We extracted the parameters from the review article to simulate the growth curve.[1]
Figure 1: The figure shows the growth curve simulation of wild type
E. coli and E. Hybrid for 8 hours. The growth curve is simulated by the Mathematical Growth Model. The glucose is 22 mmol at first and the start unit of E. coli is 0.02-gram dry weight on a 1 L container.
E. coli and E. Hybrid for 8 hours. The growth curve is simulated by the Mathematical Growth Model. The glucose is 22 mmol at first and the start unit of E. coli is 0.02-gram dry weight on a 1 L container.
From Fig. 1 above, we can see that E. Hybrid would grow better if the glucose uptake rate of E. Hybrid does not lower than wild type E. coli.
Then, we validate the model and concept by testing the experimental data.
Figure 2: The living cell of wild type E. coli and E.Hybrid for 14 hours.
Table1:The table is the error rate of growth curve prediction under 22mM Glucose.
From Fig. 2 we can see that the growth rate constant of E. Hybrid gconst is 0.44 and 0.36 [1/hour] for wild type E. coli. It increases about 22.22%. The R-square for both fitting higher than 0.97
stand for high accuracy and correspondence. We can also know the cell cycle is 1.9254 and 1.5753 [hour] for wild type E. coli and E. Hybrid and reduced to 81.82% of the original.
For the device, we also use the Intelligence Production Model to determine the refresh strategy and further increase the effect of the entire system.
Figure 3: The production potential and density of E. Hybrid for 400 hours. It would refresh for once a day and the refresh ratio is 33%, 50% and 66% under 37℃.
We can see from Fig. 3 that the refresh ratio is equal to the volume of the old culture medium divided by the volume of the new culture medium. The production potential is higher for two to one refresh ratio in two days. If the refresh
ratio is one to two, it can keep more steady in 400 hours. Above all, the model can make the device more intelligent.
Lab work
More information in Design and Experiment Result
We conducted phototrophic growth measurement to test the function of GR. From Fig. 4, we can see that GR-expressing E. coli grows faster than E. coli WT because of the positive effects on the metabolisms from the expression
of the proton pump. To know if GR can serve as an alternative for respiratory electron transport chain, we inhibited respiratory electron transport chain by adding cytochrome C inhibitor, sodium azide. From Fig. 5, we can see that
GR produces ATP in the pathway that is substantially different from the respiratory system.
Figure 4: Phototrophic growth measurement of GR-expressing
E. coli with/without sodium azide addition
E. coli with/without sodium azide addition
Figure 5: Phototrophic growth measurement of GR-expressing
E. coli under sodium azide(0.001%)
E. coli under sodium azide(0.001%)
Then, we want to know whether target protein expression will increase with our GR-expression system which makes E. Hybrid serves as a fundamental strain for bioengineering success, so we incorporated the RFP expression. From Fig. 6, we
can see that RFP expression in GR-expressing E. coli is better than the one without GR expressing system.
Figure 6: RFP expression in GR-expressing E. coli
(*: p value<0.05/**:p value<0.01/***:p value<0.001/****:p value<0.0001)
(*: p value<0.05/**:p value<0.01/***:p value<0.001/****:p value<0.0001)
From these two experiments, we learn that with the additional ATP produced by GR, GR-expressing E. coli can not only enhance its growth but also increase the target protein expression. To sum up, we proved that E. Hybrid can incorporate
with different target proteins for various applications.
Demonstration
Featuring properties of E.Hybrid in our device, we want to build a possible prototype to simply demonstrate the feasibility of E.Hybrid.
Light and heat from the sun is an everlasting gift from nature, if we can use it properly. Recently, our society has successfully transferred them into electricity, but its efficiency is not enough. With same thoughts, we want to induce the
concept into synthetic biology.
So we create E.Hybrid – E. coli with GR and its own incubating system: SSIS – Synthetic Smart Incubation System.
Demo of device
We use the indoor light to incubate due to biosafety, and continuously record the parameter we get (see Fig. 7 below).
Figure 7: Energy system minimum valuable prototype
Video 1: Energy system operating
Video 2: Incubation system operating
We compared the condition with experiments of wet lab and the prediction from model, we found it is fit and the the incubating system is work.
Figure 8: Analysis of incubating result
Reference
- Claassens, N. J., et al., Potential of proton-pumping rhodopsins: engineering photosystems into microorganisms.", 2003