Team:SCUT/Project/System Construction/Co2 Fixation

Design

Apparently, when happen in a limited space, reactions accelerate owing to bigger probability of collisions between molecules. To utilize this nature, we recruited scaffold proteins. In spite of the fact that there are endogenous scaffold proteins in S.cerevisia, we used GBD, SH3 and PDZ, three exogenous scaffold proteins so as to avoid competing with some endogenous metabolic pathways. By this way, we aimed to minimize the negative effect on metabolisms of host cells and hence to optimize our system.

We construct different radios of scaffold proteins, GBD(x), SH3(y) and PDZ (z), of which can combine with PRK, RuBisCo and CA. By altering the number of the domains, we hope to obtain the most appropriate scale of scaffold proteins and get the best result. In view of the high efficiency of PRK, we chose x=1 at the first attempt (#GBD: #SH3: #PDZ, x: y: z), and will choose the optimal radio or construct other ratios according to the output of ethanol.

The design of scaffold protein is based on the Tom family. As shown in the figure, there is a protein complex called Tom family located on the outer mitochondria membrane. Tom family which contains Tom20, Tom22, Tom40, Tom5, Tom6 and Tom7 subunits can help the protein synthesized in the cytoplasm with presequence enter into the mitochondria. Because Tom22 is anchored to the outer membrane by its hydrophobic segment in the middle of the sequence, exposing the N-terminal and C-terminal domains to the cytosol and the outer membrane, intermembrane space(IMS). Thus, we decide to design fusion protein containing target proteins and Tom22. We hope we can successfully build up the scaffolding protein binding on the outer mitochondria membrane and help finish the rest of jobs.

It is stated in the background that increased expression level of PRK would bring bad effects to the host cells, which means a something should be taken into the construction of PRK recombinant molecule for regulating its expression level. Hence, we recruited a commonly used inducible promoter, pGal1 for initiation of translation. In the meantime, we quantified Gal1 promoter activity to find out the optimal point/range of induction dose (shown in Quantification of Promoter Activities).

Construction

The five genes encoding these six enzymes were cloned into two different plasmids-----YEplac181 and YEp352, which can coexist in one cell.Considering our multiple-enzyme system, it is necessary to use those compatible plasmids. Most genes were synthesized by Genewiz: Hbd, Crt, AdhE2 (C. beijerinckii), and Ccr ((Streptomyces collinus). While S. cerevisiae gene: Erg10 were cloned from genomic DNA. And all enzymes were under control of GAL1 promoter with CYC1 or ADH1 terminator. We combine not only our enzymes PRK, RuBisCo and CA with the plasmid Yeplac181 but also the GBD,SH3 and PDZ with the plasmid Yep352.Both of them will transform into one cell. We've construct different scales in order to find out the best scale to fit our pathway enzyme such as PRK, RuBisCo and CA

These are constructions of our pathway and scaffold protein.

Figure 4 | We made our construction in plasmid Yeplac181 and Yep352.The construction of PRK, RuBisCo and CA were built up in the plasmid Yeplac181. Moreover, the FP + ligand was also constructed in the plasmid Yeplac181.Yeplac181 and Yep352 , which can coexist in one cell.Considering our multiple-enzyme system, it is necessary to use those compatible plasmids so we added the GBD+SH3+PDZ scaffold protein sequence in the plasmid YEp352.

Fiugre 5 | The SCUT A is the pathway that the RuBisCo contains the molecule chaperone in contrast with the SCUT B which lacks the molecule chaperone. The SCUT 1-1-Y means the construction includes the molecule chaperone and can fix on the outer mitochondria membrane successfully. It also means that 1-1-Y includes two device, one is the GBD, SH3, PDZ scaffold protein with different scale(1-1 means the SH3:PDZ ) ,the other is the pathway of the carbon dioxide fixation whether it owns the molecule chaperone. On the contrary the 1-1-N is the oppisite.

Figure 6 | At the pre-experiment, to test out whether CA can make a difference or not, we had two constrast recombinant groups, one(group Ⅰ) consisting of PRK-RuBisCo(in YEplac181) and GroES-GroEL(in YEp352), the other（group Ⅱ） consisting of PRK-RuBisCo-CA(in YEplac181) and GroES-GroEL(in YEp352) transfered into competent yeast cells. The colonies were first cultured in YPD media and then cultured in media containing galactose as carbon source, during which the yeast underwent a fermentation process and samples were taken out at a certain time for analysis. As shown below, at the beginning of the fermentation process, ethanol concentrations of contrast groups are higher than that of wild type cells（group 0）. With time going by, for group 0 and group Ⅰ ethanol concentrations increased first and then decreased. For group Ⅱ , on the contrary, ethanol concentration decreased first and then increased.

Leading Peptide Testing

We construct the BFP, YFP, and GFP fusing with the GBD, SH3 and PDZ ligand in order to check whether the ligands with targeted protein can bind to the domains of GBD, SH3 and PDZ as well as the scaffold protein can position on the outer mitochondria membrane or not. If it works, the fluorescent protein can gather in the same site in the cell.

Figure 7 | This is the Co-localization of BFP, YFP and GFP in mitochondria. We made he fluorescent protein fusing with the GBD, SH3 and PDZ protein ligand so that they can bind to the domains of GBD, SH3 and PDZ protein(GBD:SH3:PDZ=1:4:3). Now we see that in picture 1, it's obviously that the BFP is much brighter in somewhere than other site. And the picture 2 is the bright filed of our cell. Similarly, there is no doubt that in pictrue 3 and 4, the GFP and YFP has been gathered in several places of the cell. Picture 5 is the same cell that we dye the mitochondria. In picture 6, we gather all the fluorescent protein and the dyed mitochondria in the same image. As shown in the picture, BFP ,GFP and YFP has been overlapped with each other and they gather in same place of the cell. So, In short, We can come to a conclusion that With the help of Tom22 , we've successfully construct the scaffold protein on the surface of outer mitochondrial  membrane.

Figure 8 | The same results come from cotransformation of fluorescence protein and scaffold proteins in different ratio (GBD:SH3:PDZ=1:4:3 and GBD:SH3:PDZ=1:2:3) shows successfully colocalizing.

References

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