Team:Kyoto/Discussion

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Table of contents

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

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 accumulate Na+ inside the cell, expressed the chaperones, produced the compatible solute to reduce the damage the cells receive from high salt concentration, and added various genes for sequestering Na+ in vacuoles to the collection of BioBrick parts. With the help of mathematical modeling, we optimized the system, and eventually produced a yeast that retains averagely 80 mM of Na+ into the cell. Also, we performed the model experiment that this device really absorbed Na+ from the solution and demonstrated the decrease of Na+ in solution by it.

2)Performance and application of "Swallowmyces cerevisiae"

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

Our pilot experiments showed the repertoires of proteins nonspecifically interacting with GFP are different between a solution with a salt concentration of 1000 mM and a solution with 500 mM. 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, the yeast can grow even in the solution of NaCl concentration 1000 mM (inhibition of the growth is seen though). However, in order to accumulate Na+ inside of yeast, it is necessary to turn off these pumps. As seen in this study, yeasts of such strains become 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 the present situation.

In this experiment, mangrin and ZrGPD1 were used and had the effect of increasing salt tolerance. In this project, regarding these, 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 and 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.[1] 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.

Secondly, 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. It is known that this factor, when expressed in Arabidopsis thaliana, causes remarkable salt tolerance, for example, from facts confirmed when that homologues of ice plant which is a salt tolerant plant containing it were cloned and it expressed in A. thaliana in the past. 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 this way, a much more efficient biological desalination method than current levels may be accomplished by not only improvement of individual elements but also utilization of mathematical models and repetition of combination optimization.

3)Biocontainment system

The conjugations of cell or between cells using SdrG-Fgβ binding was hardly visible in this project. This may be because, as we mentioned in the results, we did not give these devices enough time to combine. We need to combine them 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-FgBeta to add new tools. And, in addition to the SdrG-FgBeta system, there are other known systems known as pairs of proteins that induce cooperative binding. The most famous are the pair of Spy-tag and Spy catcher. This 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.

With respect to Escherichia coli, a paper that "Assembling E. coli with each other using nanobody's surface display" has been reported recently.[2] We succeeded in fixing yeast surface-displayed flag tag fusion protein, with magnetic beads conjugated with anti-Flag antibody this time. By replacing the same display system with a pair of nanobody-antigen described in this document, it is expected that the cell assembly of budding yeast can be freely controlled.

Also, if the aggregation system is to be actually used as a method of biocontainment at the actual sites, indirect assemblies using something small molecules may be more effective than yeasts do not directly assemble. If all of yeasts expressed the nanobody targeting the same small molecule, it will be able to aggregate yeast efficiently by the administration of a small molecule to the population.

Reference

• [1]Hou,Lihua Wang,Meng Wang,Cong Wang,Chunling Wang,Haiyong (2013) Analysis of salt-tolerance genes in zygosaccharomyces rouxii, Applied Biochemistry and Biotechnoloogy 1417-1425
• [2]C.Mcmahon, A.Baier, R.Pascolutti et al. (2018) Yeast surface display platform for rapid discovery of conformationally selective nanobodies Nature Structural & Molecular Biology Vol.25