Wet Lab

Read excellent literatures about synthetic biology and projects on iGEM Official Website.

Wet Lab

We discussed with our instructors about the project designing and decided to improve Saccharomyces cerevisiae surface display technology. Then we read iGEM projects about Saccharomyces cerevisiae surface display.

Wet Lab

We had a brainstorm about yeast surface display.

Wet Lab

After discussion, we set our initial project theme that displaying human tubulins on the surface of Saccharomyces cerevisiae. Moreover, we read more literatures about tubulins.

Wet Lab

We visited Mr Meng who is the doctor of Chinese Academy of Sciences and discussed our project with him. Through the discussion, we found it difficult to keep tubulins lasting polymerization in vitro. So we attempted to look for more stable materials. At last, we found flagellin which can polymerize spontaneously in vitro.

Wet Lab

Our experiment was discussed and designed. We divided the display system into two parts: the display modules and the secretory modules. With the special structure of surface display system, we can display the particular subunit onto the Saccharomyces cerevisiae surface as a linkage site and secrete other subunits into the extracellular environment to create a subunit-rich surroundings. With the optimal concentration of subunits, GTP and other ions, those subunits could reconstitute into microtubules and flagellum. In this way, we can finally display the microtubule and flagellum on the cell surface. And then, we can use this upgraded system to facilitate small molecules screening.

Dry Lab

Build a mathematical model to test the theoretical possibility of polymerization for tubulins and flagellins in vitro. Try to find the most appropriate length for linker to improve the possibility of polymerization.

Wet Lab

In microtubule part, we intended to construct 8 vectors, they are pYD1-α、pYD1-β、pYCα-α、pYD1-mCherry(positive control)、pYCα-β、pYCα- mCherry-α、pYCα-β-mGFP、pYCα-mCherry(positive control).

Search DNA sequences (flic、PETase、XynA、BG、EG、CBH)

Dry Lab

We studied the mechanisms of tubulin and flagellin polymerization and discussed the modeling problem.

Wet Lab

Obtain the target genes sequence(α-tubulin、β-tubulin、mCherry、GFP)and plasmids (pYD1:used to display, pYCα：used to secrete). We designed primers by using snapgene and sent them to the company for synthesis.

Design and optimize DNA sequences of flagellin and enzymes(PETase、XynA、BG、EG、CBH). Send them to America for synthesis.

Dry Lab

We establish models using statistical mechanics describing the process of tubulin polymerization in vitro.

Wet Lab

Plasmids were digested by enzymes, and we had an agarose gel electrophoresis to prove true.

We used PCR to amplify target genes (α-tublin、β-tublin、mCherry、GFP) and use agarose gel electrophoresis to prove true. Then, we utilize infusion technology to ligate target genes with pYD1 or pYCα，and had an agarose gel electrophoresis to prove true. Moreover, we transform vectors we constructing into competent Escherichia coli cells(DH5α). Screen on LB plate(added Ampicillin). Send to the company to make sure the sequence.

Dry Lab

We used computers to mimic the process of binding ligands and receptors of tubulin.

Wet Lab

As sequencing results show, we had successfully constructed pYD1-α and pYD1-mCherry. We continued constructing the rest plasmids ( pYD1-β、pYCα-α、pYCα-β、pYCα- mCherry-α、pYCα-β-mGFP、pYCα-mCherry).

Dry Lab

Build a mathematical model to show the process of polymerization for flics in vitro.

Wet Lab

According to the growing curve we measured, Saccharomyces cerevisiae competent cells were made up, and we attempted to transform recombined plasmids in it using chemical transforming method.

Continue constructing plasmids.

Wet Lab

We successfully constructed recombined plasmids(pYCα-α、pYCα-β、pYCα- mCherry-α、pYCα-mCherry). We constructed pYD1-β once again.

Continue transforming recombined plasmids in Saccharomyces cerevisiae (EBY100 & invsc1) competent cells using chemical transforming method.

Wet Lab

Obtain the synthetic DNA flagellins and enzymes including PETase、XynA、BG、EG and CBH.

We prepared for the Final Exam so we stopped the experiments.

Wet Lab

pYD1-β was constructed successfully.

Plasmids were digested by enzymes, and we had a gel electrophoresis to prove true.

To get recombined plasmids(pYCα-PETase、pYCα- XynA、pYCα- BG、pYCα- EG、pYCα-CBH) containing corresponding enzyme, we fused flic-N respectively with five enzymes(PETase、XynA、BG、EG、CBH) through PCR to get a part of target gene. Then we infused the part of target gene with flic-C and plasmids digested by enzymes. These plasmids can express secretory proteins. Screen strains on LB plate (added Ampicillin). Send sequencing.

Recombined plasmid pYD1-flic-Mgfp is constructed in the same way with pYCα-PETase、pYCα- XynA、pYCα- BG、pYCα- EG、pYCα-CBH.

We tried electronic transformation way to transform plasmids into yeast competent cells due to the fact that we always failed in chemical transformation way.

Dry Lab

After collecting data, we constructed a three-dimensional model of tubulin polymerization, in which the independent variables were temperature, concentration, and energy GTP.

Wet Lab

Send recombined plasmids including pYD1-flic-mGFP、pYCα-PETase、pYCα- XynA、pYCα- BG、pYCα- EG and pYCα-CBH sequencing.

We succeeded in electronic transformation way, transforming pYD1-mCherry into yeast competent cells and screening on SD plate (added Leu).

Wet Lab

We transformed pYD1-mCherry into yeast competent cells EBY100 in electronic transformation way and screened on SD plate (added Leu), expanding culture.

Plasmids ( pYCα-PETase、pYCα- XynA、pYCα- BG、pYCα- EG、pYCα-CBH )had successful sequencing.

Reconstruct pYD1-flic-Mgfp.

Dry Lab

We analyzed the structure of tubulin and flagellin by computer.

Wet Lab

We transformed pYD1-α、pYD1-β into yeast competent cells EBY100 successfully.

We constructed pYD1-flic-mGFP successfully.

We transformed pYD1-flic-mGFP into yeast competent cells EBY100 in electronic transformation way and screened on SD plate (added leu), expanding culture.

Wet Lab

We transform plasmid pYD1-mCherry (positive) into Saccharomyces cerevisiae EBY100 and observe that the extracted protein have red fluorescence. The expression of target protein in EBY100 is successfully. A strong reducing agent DDT is added to the cell culture medium of EBY100 to break the disulfide bond. Then the target protein mCherry displays on the Saccharomyces cerevisiae surface is separated from Saccharomyces cerevisiae EBY100, the protein in the supernatant is extracted and the fluorescence is observed.

Wet Lab

We transformed pYCα-α、pYCα-β、pYCα- mCherry-α、pYCα-β-mGFP、pYCα-mCherry (positive) into yeast competent cells invsc1 that was cultured on SD plates including leucine, tryptophan, histidine to screen.

A strong reducing agent DDT is added to the cell culture medium of EBY100 to break the disulfide bond. Then we extracted the Saccharomyces cerevisiae EBY100 which were successfully transformed in plasmids pYD1-flic-mGFP. The protein in the supernatant was extracted and use coomassie blue staining to prove true. The protein was successfully extracted and the coomassie blue staining experiment succeeded.

Wet Lab

We successfully transformed plasmids(pYCα-α、pYCα-β、pYCα- mCherry-α、pYCα-mCherry) into invsc1.

Wet Lab

Saccharomyces cerevisiae that was transformed in plasmids(pYCα-α、pYCα-β、pYCα- mCherry-α、pYCα-mCherry) successfully expressed the target protein.

Wet Lab

Through coomassie blue staining, target proteins expressed by pYD1-α、pYD1-β were successfully tested . Western Blot is used in chemical coloration to confirm the target protein.

We construct models to simulate the actual working state of Tubulin and Flagellum.

Wet Lab

The effect of substrate DAB staining on protein detection is not ideal in Western blot. We attempted to use a more sensitive ECL color-developing method and achieved successfully.

Saccharomyces cerevisiae that was transformed in plasmids(pYD1-α、pYD1-β) successfully expressed the target protein in cells.

Western Blot in ECL color-developing method confirmed the target protein was expressed.

Wet Lab

Through coomassie blue staining, target proteins expressed by pYCα-α、pYCα-β、pYCα- mCherry-α、pYCα-β-mGFP、pYCα-mCherry (positive) were successfully tested . Western Blot in ECL color-developing method confirmed the target protein.

Wet Lab

We transformed pYCα-α、pYCα-β、pYCα- mCherry-α、pYCα-β-mGFP、pYCα-mCherry (positive) into yeast competent cells invsc1 that was cultured on SD plates including leucine, tryptophan, histidine to screen.

Wet Lab

Through coomassie blue staining, target proteins expressed by pYCα-PETase、pYCα- XynA、pYCα- BG、pYCα- EG、pYCα-CBH were successfully tested . Western Blot in ECL color-developing method confirmed the target protein.

Protein purification

Plasmids pYC, -PETase, pYC, alpha - XynA, pYC - - BG, pYC - - EG and pYC - -CBH were successfully transformed into Saccharomyces cerevisiae invsc1.