Team:Kyoto/Result
In starting the experiment, we conducted an experiment demonstrating how the salt concentration of the solution actually affects the protein-protein interaction by using the model protein. In this example, we chose GFP(BBa_E0040), which is commonly used in many experiments as a model protein.
TDH3 promoter and CYC1 terminator were added to both ends of ORF and cloned into pRS316 which is a shuttle vector of S. cerevisiae and E. coli. The resulting plasmid was transformed into wild-type yeast strain BY4741 to overexpress GFP in yeast. As a comparative experiment, yeast expressing RFP(BBa_E0010) with the same set of promoter and terminator was used.
First, photographs of pellets recovered from the culture medium of yeast cells used in this experiment are shown.
Figure1. A picture of yeast expresses RFP or GFP
As can be easily seen, the yeast pellet overexpressing GFP was colored in a pale yellow color while the yeast overexpressing RFP was colored in a thin red color. From this, it was confirmed that both GFP of BBa_E0040 and RFP of BBa_E0010 can be expressed in large amounts in yeast cells without changing the codon and that the expression level thereof is so large as to be visually observed under visible light without breaking the yeast.
In order to investigate yeast proteins interacting with GFP, an experiment was conducted to purify expressed GFP protein from yeast lysate using immunoprecipitation. Anti-GFP nanobody (GST fusion protein on Glutathione sepharose beads) was used for sedimentation, and the obtained precipitate was visualized by SDS-PAGE and subsequent Silver Stain.
This yeast was placed in a mortar, liquid nitrogenized and crushed, and a lysate was prepared with a buffer having a salt concentration of zero. The color from the fluorescent protein was clearly transferred to the supernatant by this treatment in both proteins. Figure2 shows the results of electrophoresis of GFP pull down after adding Sepharose bead conjugated with anti-GFP nanobody to this lysate.
Figure2. Immunoprecipitation of GFP from Yeast lysate
As shown in Figure2, the band of ① is GST-GFPnb, and the band of ② (5-8) is GFP. GFP is clearly immunoprecipitated by GST-GFPnb. Furthermore, since the band intensities of ① and ② are equal, it is understood that GFP and GFPnanobody are linked at about 1: 1. This indicates that the binding between this GFP-GFPnanobody is strong and GFP is sufficiently contained in the lysate.
In the band of ③, bands are seen at low salt concentration, and it can be confirmed that the band becomes thinner as the salt concentration becomes higher. This is thought to be due to the nonspecific interaction between nanobody and the protein contained in the lysate under low salt concentration. In addition, the band of ④ of 8 lanes is a band that can be seen only at 1000 mM and in the presence of GFP. This is thought to be due to the interaction between GFP and protein in lysate generated under high salt concentration condition. These suggest that salt concentration can influence the three-dimensional structure of protein and its interaction.
From next section, we will show how to prepare yeast which adjusts salt concentration in solution.
2-1) To inhibit Na+ efflux
In order to make yeast that effectively intake the salt from the environment, we aimed to reconstruct in yeast Na+-isolation system of salt-resistant plants, which sequester Na+ into their vacuoles. We first tried to knockout NHA1 and ENA1, the critical components of Na+ efflux of budding yeast.
Since there is no ENA1 knockout strain in the Yeast Knockout Collection, we amplified hygromycin resistance cassette from pFA6a-hphMX6 plasmid and tried to knockout genomic ENA1 gene utilizing homologous recombination. We transformed PCR products into wild type yeast and obtained hygromycin-resistant colonies. Genomic PCR from obtained colonies successfully identified the strains that have desired mutation. Using the same method, we successfully made ΔENA1ΔNHA1 strain from ΔNHA1 strain.
Moreover, we asked Dr. Uozumi in Tohoku University to provide us with G19 strain that lacks all of ENA1 family genes(W303 yeasts have a cluster comprised of ENA1-ENA2-ENA3-ENA4). Although he kindly gave us G19 strain, we also introduced ENA1,2,5 knockout strain, (they are equivalents of ENA1-4 in W303 yeasts) to ΔNHA1 strain, due to the limited repertoire of selection markers that G19 strain has.
Finally, we got ΔNHA1 / ΔENA1ΔNHA1 / ΔENA1 / ΔENA1,2,5.
All genes we disrupted so far encode Na+ or K+ transporters that are responsible for Na+ export from cytoplasm.
Therefore, disruption of any of these genes would lead to decreased export efficiency of excessive salt. Some researchers reported that yeast that lacks these genes showed increased NaCl-sensitivity. To compare salt-resistance of knockout strains we prepared, we performed in vivo spotting assay.
Figure3. Spotting assay of KO yeast strains
Figure3 shows representative results of spotting assay. With 200 mM NaCl in YPD, wild type strain formed large colonies after 2 days of incubation. In contrast, G19 strain and ΔENA1,2,5ΔNHA1 showed little or no colonies, indicating dramatical increase of salt-sensitivities of these strains. Under the same condition, Zygosaccharomyces rouxii, an yeast strain known to show high salt-resistance (as they have been used to make soy sause in Japan), made large colonies. This results encouraged us to integrate genes of Z. rouxii to our device.
2-2）To increase salt-resistance of yeast
The result we described above indicate that we successfully disrupted proper genes for our purpose, and thus mutants could not export Na+ from their cytoplasm, which is favorable to make salt-absorbing yeasts. However, from these results, we also noticed serious problem that our project has.
As long as we use the mutants that absorb salt, we have to face the problem of salt-sensitivity that our device has. If we could sequester Na+ in the vacuole of yeast as we initially planned, salt-resistance might be recovered. However, even the yeast equipped with Na+ sequestration system would suffer from high salt condition to some extent. Hence, we concluded that we have to give yeast salt-resistance to overcome this problem.
In order to make salt-resistant yeast, we took three approaches; to sequester Na+ into vacuole by active transportation as we initially planned; to prepare compatible solutes to mitigate cell stress caused by osmotic pressure; and to minimize the alteration of the higher order structures of proteins, which is caused by high Na+ concentration, by efficiently expressing chaperons.
2-3) How much of the Na+ can be absorbed by the device
With the assistance and advice of many experts (link for SLiCE etc.), we successfully made the parts we describe here (link for parts). We then sub-cloned these genes into the plasmid that could be used in budding yeast and transform them into yeast. We used high copy number plasmid derived from 2-micron as an expressing vector. We also used low copy number plasmid derived from CEN plasmid because expression of some exogenous genes from high copy plasmid might lead to cytotoxicity (e.g. growth inhibition) to yeast. As overexpression of exogenous membrane protein may lead to stress response such as ERAD, we have to be careful about expression of membrane protein, main components of our parts. Therefore, we have to carefully tune the expression level. Yeast harboring AVP1, AtNHXS1, SseNHX1, and mangrin in high copy plasmids showed significantly low growth rate (Table XXX). Especially, yeast harboring mangrin gene in high copy plasmid could not even form a colony.
We cultured various mutants we made in the media containing 400 mM NaCl and measured the amount of Na+ uptake by XXX method during the yeast growth (from OD=0..5 to OD=1.0). We show average Na+ concentration of yeasts below.
First, we measured Na+ uptake by mutants that do not harbor any plasmids. As shown in fugure XXX, even the wild type strain presented Na+ uptake to some extent. As we expected, the strains that lack the elements for NaCl export, such as NHA1 and ENA1, retained more Na+ than wild type in their cytoplasm. Among them, G19 strain, which lack NHA1 and all of the ENA1 family, and strain, which mimics G19 strain, showed especially high Na+ concentration. These results support our NHA1ΔENA1ΔENA2ΔENA5Δhypothesis that these elements are main components of NaCl export and indicate that NHA1ΔENA1ΔENA2ΔENA5Δ should be the chassis of Swallowmyces cerevisiae.
Interestingly, K+ concentrations of the same sample sets were greatly different from those of Na+. Again, NHA1ΔENA1ΔENA2ΔENA5Δ that we made showed a remarkable feature. Although this strain took in the greatest amount of the Na+, it took in the smallest amount of the K+ among 6 strains compared, showing that its selectivity of Na+/K+ uptake is the best among them.
Next we conducted a similar experiment on NHA1ΔENA1Δ strain which expressed the parts we constructed and tested the quality of plasmid on salt absorption.
The result is shown in the figure. We could understand that host strain that only posses empty vector takes up Na+ until the mean of intracellular concentration becomes about 25mM. (NaCl concentration of culture medium is 400 mM). From the plasmid, various factors were expressed and the amount of NaCl absorbed was measured and compared each other. As a result, the intracellular retention of NaCl was most efficiently observed in the strain expressing SseNHX1, and the strain in which AtNHXS1 was expressed absorbed NaCl at almost the same level, ranking in the second.
These factors are expressed in vacuoles and they transport H + in vacuoles to the cytoplasm and transport cytoplasmic Na + to vacuoles. In simplest model is like this; Saccharomyces cerevisiae that expressed these factors behaves just as like of salty tolerance plants that store Na + taken from extracellular into vacuoles, so the amount of Na + that can be stored by the whole cells remarkably increased.
These factors showed the maximum uptake of NaCl when expressed from low copy plasmid, and when expressed by multicopy plasmid, the effect of both genes was greatly attenuated.
The AVP1 gene is a Pyrophosphate-driven H + pump localized on the vacuolar membrane and plays a major role in the enrichment of H + in vacuoles. There is a report that by expressing this gene derived from At will improve salt tolerance in budding yeast.
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This is interpreted as an increment of the H+ concentration in the vacuole, which promotes the relatively inactive endogenous NHX1 function of Saccharomyces cerevisiae and isolates cytoplasmic Na+ in vacuoles. We believe that the increase in the total Na + content in the cells is due to the expression of the AVP1 gene and this is caused by the same mechanism as this.
Interesting results were also obtained for AtHKT1. In contrast to other factors, this factor has been reported to make the expressing yeast highly sensitive to salt. However, in this NaCl absorption assay, it was found that it contributes to an increase in the average Na+ concentration in yeast as well as other factors. This suggests that AtHKT1 is involved in afflux of NaCl and anchoring to yeast by a different mechanism from other localized pump expressed on vacuolar membrane. It has been reported that AtHKT1 is a co-transporter of Na+/ K+, it is localized on the cell membrane and seems to be directly involved in an influx of Na + from external solution (Uozumi 2000).
Another interesting example is gene mangrin and ZrGPD1. The former is a small peptide that works like a chaperone and initially, we were expecting them to protect host yeast from damage caused by salt. We have never thought that mangrin itself will impact on absorbing NaCl. However, when we did an experiment, it seems that mangrin will encourage the absorption of NaCl very strongly. Similarly, ZrGPD1, which is involved in the production of glycerol as a compatible solute, was also introduced to protect yeast from osmotic stress. In this case also, although the mechanism is unknown, uptake of salt appearance seems to be involved in by some mechanism, and salt uptake of host yeast increased by an introduction of overexpression plasmid.
As an aim to construct Swallowmyces cerevisiae, we have successfully made several devices that absorbs NaCl from a culture. Furthermore, we proved and demonstrated that each part actively transports NaCl in the medium into the cell by a different mechanism.
2-4) How much of the Na+ can be absorbed by the device
Our primary goal is to construct the device which assists the function of the various synthetic biological devices used in the same test tube by absorbing the Na + contained in the solution and modulating the salt concentration in the test tube. As indicated above, the construction of yeast which actually absorbs and reduces NaCl in the medium has been completed.
However, during Human Practice's activities, the initial projects underwent major changes due to two points (link). The first change is the extension of future goal. We thought that this biological desalination system could be applied outside the laboratory for measures against salt damage that is a big social problem. the biological desalination system can be applied outside the laboratory for measures against salt damage that is a major social problem.The second problem is the problem of biocontainment that cannot be avoided when using genetically modified yeast outside the laboratory.
As shown in the figure (link) of Human Practice, a column type container for fixing and using yeast has already been put to practical use. Originally we planned to use this container as it was. However, we noticed that this system is inefficient as it is. There is a problem that the column cannot be reused because it is difficult to replace the internal yeast. Yeast that has absorbed a certain amount of NaCl needs to be devised so that the incorporated salt does not return to the environment, such as removing it from the column and incinerating it. Because of the system utilizing live yeast, the user must pay close attention so that small yeast will not leak into the environment.
In this situation, as a way to simplify the handling of yeast as much as possible, we worked on the creation of yeast artificial aggregation induction system. Specifically, we use a very strong interaction (paper) between SdrG and FibBeta. If yeast's surface display system can be used to present these two proteins on different yeast surfaces, yeasts will be tied together by strong interactions and form large cell clusters. Such a cell mass can be handled easily in handling rather than handling a single small yeast cell and it is considered to be effective for preventing leakage of genetically modified yeast to the natural world.
The binding domain of SdrG and the artificial sequence which becomes the stalk region were fused with FgBeta and fused with the anchoring domain of Sed1 and presented to the cell surface. Plasmids expressing GFP or RFP (used in the above immunoprecipitation) were simultaneously introduced into the same yeast so that the yeast was colored, so that each cell could be distinguished.
Both of them were singly or mixed and kept warm, after which the association of both occurred, diluted and observed with a fluorescence microscope. The results are shown in the figure.
In order to see the interaction between SdrG and FgBeta created in vitro, these proteins were synthesized in a cell-free translation system and pulled down with a His tag fused to SdrG and a Flag tag fused to FgBeta It was. The results are shown in the figure. As shown in the figure, SdrG was synthesized in a cell-free translation system and showed a clear band of the expected size. Pull-down by Ni-NTA is also shown, indicating that the target tag sequence is also working. However, it appeared that FgBeta was hardly expressed in cell-free translation system. Since the monoclonal antibody used for immunoprecipitation was strongly detected at the time of Western blotting and became noise, it can not be concluded that translation products do not necessarily exist. However, both Rabbit Reticulocyte Lysate and Wheat Germ Extract showed the same tendency (both SdrG can be observed but FgBeta cannot be observed), so FgBeta shows an unstable sequence in lysate It may be thought that it may be included. Probably due to this reason, coprecipitation of FgBeta and SdrG could not be observed in either lysate.
From another experiment we could obtain evidence that FgBeta is actually expressed in the cell and displayed on the surface of the yeast.
FgBeta contains a Flag sequence in its ORF. This sequence is inserted outside the stalk structure of extracellular domain (an artificial sequence of length 649 aa is inserted to avoid steric hindrance on the cell surface). Yeast displaying FgBeta on the cell surface was captured with magnetic beads coated with anti-Flag antibody, and after washing with PBS, the bound cells were observed with a microscope.
The results are shown in the figure. As expected, yeast containing FeBeta (Flag) bound very well to anti-Flag antibody beads. It was observed that yeast with many RFP signals was bound to the washed beads. In contrast, when yeast displaying SdrG (His 6) was used in the same manner, yeast with almost GFP signal did not settle. This indicates that anti-Flag antibody beads do not randomly precipitate yeast at all, but rather specifically recognize and settle the Flag sequence displayed on the cell surface. From the above results, the following was clarified.
(1) FgBeta (Flag) was appropriately displayed on the surface of yeast cells by fusion with Sed1 anchoring domain.
(2) Only specific yeasts could be immobilized on magnetic beads using the antigen placed on the surface.
SdrG (His 6) was similarly displayed on the cell surface by Sed 1 anchoring domain, but SdrG (His 6) expressing cells could not be recovered by recovery with His-trap magnetic beads. This may be due to the reason that the presented His6 sequence is in a situation that does not allow access of magnetic beads due to steric hindrance, or because SdrG (His 6) has a lower expression level per cell.
In addition, mixture of Fgβ(FLAG) expressing cells and SdrG (His 6) expressing cells was captured with magnetic beads coated with anti-Flag antibody. Figure shows significant decrease in number of captured cell compared to Figure(-1). We cannot assume what is going on, but it may be consequent of interaction between Fgβ and SdrG.
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