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| − | + | In addition, to finally realize our design, the yeast need to ferment on xylose only. Therefore, we use the SBA-Biosensor to detect the ethanol content in the medium, which can convert the reaction of immobilized enzyme to electrochemical signal and help make a curve reflecting the ethanol change in the culture condition. | |
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Revision as of 03:47, 25 October 2017
Demonstrate
Basic fermentation
Our basic fermentation part derives from our local environmental problem, the outbreak of Enteromorpha along the coastline in Qingdao, so it is natural that we would eventually go back to the origin and try to solve the real world problem after validation of design concept in the lab. We aim to make use of Enteromorpha residue, where there is no trehalose left because it is the easiest to extract. Therefore, all we need to do is to deal with the cellulose and hemicellulose left in the residue.
And we do treat our Enteromorpha powder with enzymes first and yeast later. The successful survival of the recombinant yeast strains that can use either xylose or cellubiose as the only carbon source can fully prove the feasibility of our designed pathway.
Along with the proof of our concept, we validate the upstream pathway from real algae powder, which has exactly the same constituent as Enteromorpha residue, and we can say that our idea can apply to real-world problems!
Enteromorpha pretreatment
We treat the Enteromorpha powder (residue) with 0.2% H2O2 to remove the lignin then cellulose to produce xylose & cellubiose.
Figure 1. Enteromorpha Powder
Figure 2. Treat the residue with 0.2% H2O2.
Figure 3. Enzymatic hydrolysis solution of Enteromorpha fiber
Pretreatment validation
After that we detect the existence of them with HPLC. The peak appears at the same point suggesting that they are the same substance. In other words, we successfully proved that the downstream product of Enteromorpha powder after pretreatment contains mainly xylose and cellubiose.
Figure 4.Result of standard cellubiose.
Figure 6.Result of standard xylose.
Figure 5.Result of our sample (the doublet indicate there is impurity in our sample).
Figure 7.Result of our sample (the smaller peak indicate there is impurity in our sample).
Yeast A that Ferment xylose
We use pYC230 provided by our PI as our backbone and integrate xylosidase gene xyl1 and xyl2 through Gibson.
Figure 8.Result of introducing pYC230 into EBY100.
After introducing the plasmid into S.cerevisiae EBY100 and construct the xylose-utilize strain successfully, we measured the growth rate of both our recombinant strain and negative control.
The following result can well demonstrate that the strain that carries our plasmid grows much better than the strain that not.
Figure 9.Growth curve of strains of our recombinant strain and negative control.
For an immediate evidence, we need to know exactly how xylose content change in the medium. We use HPLC to detect the changing concentration of xylose, getting more data to support our idea that our cells can utilize the carbon sources as long as the concentration of xylose decrease with time.
The following chart shows the xylose content of both our recombinant strain and negative control.
Figure 10. Xylose content of both our recombinant strain and negative control.
In addition, to finally realize our design, the yeast need to ferment on xylose only. Therefore, we use the SBA-Biosensor to detect the ethanol content in the medium, which can convert the reaction of immobilized enzyme to electrochemical signal and help make a curve reflecting the ethanol change in the culture condition.
The following chart shows the ethanol content of both our recombinant strain and negative control.
Figure 11. Ethanol content of both our recombinant strain and negative control.
The result shows that we successfully constructed a xylose consuming yeast strain that produce ethanol at the same time.
In the same way, we conduct a series of experiment to confirm that our cellubiose-utilizing pathway also worked.
Our idea has been turn into real-world practice!
Mini system
We work on a mini system including standardized promoters and terminators with concise structure and powerful function in Yeast. In the process of precise experiment design, we found that redundant sequences of promoters and terminators limit large-scale synthetic biology efforts in yeasts. So we constructed a “mini” system, which contains “mini” promoters and terminators with concise sequences. This system can improve the expression level of heterologous genes compared with the common-used combination of promoters and terminators. What’s more, it can providing more potential for large-scale synthetic biology operations on yeast.
Meanwhile, we have made a standard quantitative description of it, hoping to establish a quantitative system with repeatable, strict and standard features and can be applied for various situations.
We successfully built the mini system and validated its high level of expression,and we also test in different laboratories and yeast strains to verify the system can still achieve the expected function.
Successfully constructed four sets of detection systems and imported into yeast EBY100
Figure 12.Different combinations of the Minip.Minit.CYC1p,CYC1t.
Successful validation of the mini system's expressive effect
Figure 13.Measured the intensity of excitation and emission of yecitrine, respectively 502nm and 532nm.The intensity of the four circuit from high to low is mm,cm,mc,cc.
Successful validation of the desired results in different laboratories and strains
Figure 14. QPCR the 22hour with the highest expression intensity. Error bars indicate s.d. of mean of experiments in triplicate.
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