Team:Kyoto/Discussion

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仲里　一応　訳中

• 1) Summary of our research
• 2) Performance and application of "Swallowmyces cerevisiae"
• 3) Biocontainment system
• 4)

1)Summary of our research

We worked on the construction of the yeast “Swallowmyces cerevisiae” which absorbs NaCl from solution and adjusts the salt concentration of the solution. We created a gene-disrupted strain designed to make Na+ inside the cell, expressed the chaperone and produced the compatible solute to reduce the damage the cell receives from high salt concentration. And, we added a variety of genes for taking Na+ into the vacuole to the collection of BioBrick parts. With the help of mathematical modeling optimizing the system, eventually we produced a yeast that retains averagely 80mM of Na+ into the cell. Also, we performed the model experiment that this device really absorbed Na+ from all over the solution and demonstrated the decrease of Na+ in solution by it.

2)Performance and application of "Swallowmyces cerevisiae"

Our early purpose was to assist the operation of other devices by lowering the salt concentration of the aqueous solution in the test tube in this device.

Our pilot experiments showed the salt concentration in a solution of 1000mM of the solution and 500mM, differ in the repertory of proteins interacting nonspecifically with GFP.As can be inferred from this example, if we really aim the effect of helping functions of other devices, it is worth working on the desalination from solution of NaCl concentration 1000mM with a little up from the current salt concentration range. The current system is still the first prototype, and it seems to be necessary to add various improvements after this.

There are several possible suggestions for concrete improvement.

First of all, it will be necessary to raise the halotolerance of yeast. Since wild-type yeast has multiple pumps exhausting Na+ outside, yeast can grow even in the solution of NaCl concentration 1000mM (inhibition of growth is seen). However, in order to store Na+ inside of yeast, it is necessary to turn off these pumps. As seen in this study, in such strains, yeast becomes sensitive even to solution of very low NaCl concentrations. It is necessary to improve so that such susceptible yeast can grow with maintaining the ability to take Na + from the external solution, even if the salt concentration is higher than usual.

In this experiment, mangrin and ZrGPD1 were used and had the effect of increasing salt tolerance. In the project regarding these facts, we merely expressed the sequence in nature using the constitutive promoters as it was. Searching for sequences that are more effective against salt tolerance or modifying expression levels or localization signals will leave room for further modification that leads to further salt tolerance increase. Also, ZrGPD1 acts as a compatible solute by producing a large amount of glycerol. But, in addition to this factor, there is a report that giving yeast high salt tolerance succeeded by simultaneously expressing the glycerol transporters encoded by ZrFPS1, in the past reports.(https://www.ncbi.nlm.nih.gov/pubmed/23673487）.This time we tried to clone this factor, but it did not succeed. By adding such a tool, salt tolerance of yeast may improve dramatically.

Second, there is a method of positively flowing external Na+ into the cell by utilizing a transporter on the cell membrane. Using AtHKT1 of the Arabidopsis thaliana, we observed mildly elevated Na + uptake into the cells this time. For example, homolog of the Mesembryanthemum crystallinum is a salt-tolerant plants have been cloned in the past and, about this factor, is known to result in significant salt tolerance in Arabidopsis when expressed in Arabidopsis. We tried cloning this McHKT2 this time, but we also could not make it succeed. By adding this factor to the tool box, the desalination at a higher level is expected to be possible.

Third, as a group of factors functioning on the vacuole membrane, there is a possibility of improvement. All of these are membrane proteins, and for many factors, only by changing the expression plasmid to a high copy plasmid, apparent inhibition of proliferation has been observed. Membrane proteins are synthesized on the membrane of the endoplasmic reticulum, maturated through intricate processing and folding in the endoplasmic reticulum, and transported to the target organelle (vacuole). Overexpression of a foreign gene may negatively affect cell proliferation by competing with the synthesis of important membrane proteins inherent in the cell. In addition, since genes expressed in plants are expressed in yeast, it is impossible to avoid the fact that many molecules that cause abnormality in maturation in the course of synthesis will appear. Under current conditions, there is a possibility that the protein synthesis activity of the cell may be decreased due to activation of ERAD and so on. In order to construct a more efficient system, it will be necessary to tune the delicate expression level of these related genes.

In addition to the improvement of individual elements, a much more efficient biological desalination method than current levels may be accomplished by repeating combinatorial optimization with the use of mathematics model.

The conjugations of cell or between cells using SdrG-Fgβ binding was hardly visible in this project. This may be why, as I mentioned in the results, we did not give these devices enough time to combine. We need to combine for a longer time and clarify in future experiment whether these pairs can actually be used for purpose.

This time, we tried to introduce a system of SdrG-Fgβ to add new tools, and other examples are known for protein pairs that induce cooperative binding. The most famous are the pair of Spy-tag and Spy catcher. These is a proven pair, reported to form a covalent bond each other by contact in a short time, and also included in the distribution kit of iGEM. It is important not only to stick to SdrG-Fgβ but also to try to adhere between cells by other methods. If we can introduce multiple binding pairs, we will be able to design cell aggregation by changing the selection of "handles" that are expressed for each cell.

3)Biocontainment system

4)

Reference

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