Difference between revisions of "Team:BNU-China/Notebook"

 
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         <p>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 could 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 could finally display the microtubule and flagellum on the cell surface. And then, we could use this upgraded system to facilitate small molecules screening.</p>
 
         <p>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 could 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 could finally display the microtubule and flagellum on the cell surface. And then, we could use this upgraded system to facilitate small molecules screening.</p>
 
         <h2>Dry Lab</h2>
 
         <h2>Dry Lab</h2>
−
         <p>Built a mathematical model to test the theoretical possibility of polymerization for tubulins and flagellins in vitro. Tried to find the most appropriate length for linker to improve the possibility of polymerization.</p>
+
         <p>Built a mathematical model to test the theoretical possibility of polymerization for tubulins and flagellins in vitro. </p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <div class="note-content">
 
         <div class="note-content">
 
         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
−
         <p>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).</p>
+
         <p>In microtubule part, we intended to construct 8 vectors, they are pYD1-α-tubulin、pYD1-β-tubulin、pYCα-α-tubulin、pYD1-mCherry(positive control)、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-β-tubulin-mGFP、pYCα-mCherry(positive control).</p>
−
         <p>Searched DNA sequences (flic、PETase、XynA、BG、EG、CBH)</p>
+
         <p>Searched DNA sequences (FliC、PETase、XynA、BG、EG、CBH)</p>
 
         <h2>Dry Lab</h2>
 
         <h2>Dry Lab</h2>
−
         <p>We studied the mechanisms of tubulin and flagellin polymerization and discussed the modeling problem.</p>
+
         <p>Tried to find the most appropriate length for linker to improve the possibility of polymerization.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <p>Designed and optimized DNA sequences of flagellin and enzymes(PETase、XynA、BG、EG、CBH). Send them to America for synthesis.</p>
 
         <p>Designed and optimized DNA sequences of flagellin and enzymes(PETase、XynA、BG、EG、CBH). Send them to America for synthesis.</p>
 
         <h2>Dry Lab</h2>
 
         <h2>Dry Lab</h2>
−
         <p>We established models using statistical mechanics describing the process of tubulin polymerization in vitro.</p>
+
         <p>We studied the mechanisms of tubulin and flagellin polymerization.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <p>We used PCR to amplify target genes (α-tublin、β-tublin、mCherry、GFP) and used agarose gel electrophoresis to prove true. Then, we utilized infusion technology to ligate target genes with pYD1 or pYCα，and had an agarose gel electrophoresis to prove true. Moreover, we transformed vectors we constructing into competent Escherichia coli cells(DH5α). Screened on LB plate(added Ampicillin). Sent to the company to make sure the sequence.</p>
 
         <p>We used PCR to amplify target genes (α-tublin、β-tublin、mCherry、GFP) and used agarose gel electrophoresis to prove true. Then, we utilized infusion technology to ligate target genes with pYD1 or pYCα，and had an agarose gel electrophoresis to prove true. Moreover, we transformed vectors we constructing into competent Escherichia coli cells(DH5α). Screened on LB plate(added Ampicillin). Sent to the company to make sure the sequence.</p>
 
         <h2>Dry Lab</h2>
 
         <h2>Dry Lab</h2>
−
         <p>We used computers to mimic the process of binding ligands and receptors of tubulin.</p>
+
         <p>We discussed the main modeling problem in our project.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <div class="note-content">
 
         <div class="note-content">
 
         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
−
         <p>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).</p>
+
         <p>As sequencing results show, we had successfully constructed pYD1-α and pYD1-mCherry. We continued constructing the rest plasmids ( pYD1-β-tubulin、pYCα-α-tubulin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-β-tubulin-mGFP、pYCα-mCherry).</p>
 
         <h2>Dry Lab</h2>
 
         <h2>Dry Lab</h2>
−
         <p>Built a mathematical model to show the process of polymerization for flics in vitro.</p>
+
         <p>We learned as many as possible modeling which maybe useful for our work.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <p>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. </p>
 
         <p>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. </p>
 
         <p>Continued constructing plasmids.</p>
 
         <p>Continued constructing plasmids.</p>
 +
                                <h2>Dry Lab</h2>
 +
        <p>We learned as many as possible modeling which maybe useful for our work.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <div class="note-content">
 
         <div class="note-content">
 
         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
−
         <p>We successfully constructed recombined plasmids(pYCα-α、pYCα-β、pYCα- mCherry-α、pYCα-mCherry). We constructed pYD1-β once again.</p>
+
         <p>We successfully constructed recombined plasmids(pYCα-α-tubulin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-mCherry). We constructed pYD1-β once again.</p>
−
         <p>Continued transforming recombined plasmids in Saccharomyces cerevisiae (EBY100 & invsc1) competent cells using chemical transforming method.</p>
+
         <p>Continued transforming recombined plasmids in Saccharomyces cerevisiae (EBY100 & IVSCc1) competent cells using chemical transforming method.</p>
 +
                                <h2>Dry Lab</h2>
 +
        <p>We discussed to make sure what problem we were about to solve.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <p>Obtained the synthetic DNA flagellins and enzymes including PETase、XynA、BG、EG and CBH.</p>
 
         <p>Obtained the synthetic DNA flagellins and enzymes including PETase、XynA、BG、EG and CBH.</p>
 
         <p>We prepared for the Final Exam so we stopped the experiments.</p>
 
         <p>We prepared for the Final Exam so we stopped the experiments.</p>
 +
                                <h2>Dry Lab</h2>
 +
        <p>We discussed to make sure what method we were about to use and what model we were about to build.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <p>pYD1-β was constructed successfully.</p>
 
         <p>pYD1-β was constructed successfully.</p>
 
         <p>Plasmids were digested by enzymes, and we had a gel electrophoresis to prove true.</p>
 
         <p>Plasmids were digested by enzymes, and we had a gel electrophoresis to prove true.</p>
−
         <p>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 could express secretory proteins. Screened strains on LB plate (added Ampicillin). Sent sequencing.</p>
+
         <p>To get recombined plasmids(pYCα-FliC(PETase)、pYCα-FliC(XynA)、pYCα-FliC(BG)、pYCα-FliC(EG)、pYCα-FliC(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 could express secretory proteins. Screened strains on LB plate (added Ampicillin). Sent sequencing.</p>
−
         <p>Recombined plasmid pYD1-flic-Mgfp was constructed in the same way with pYCα-PETase、pYCα- XynA、pYCα- BG、pYCα- EG、pYCα-CBH.</p>
+
         <p>Recombined plasmid pYD1-FliC-Mgfp was constructed in the same way with pYCα-FliC(PETase)、pYCα-FliC(XynA)、pYCα-FliC(BG)、pYCα-FliC(EG)、pYCα-FliC(CBH).</p>
 
         <p>We tried electronic transformation way to transform plasmids into yeast competent cells due to the fact that we always failed in chemical transformation way.</p>
 
         <p>We tried electronic transformation way to transform plasmids into yeast competent cells due to the fact that we always failed in chemical transformation way.</p>
 
         <h2>Dry Lab</h2>
 
         <h2>Dry Lab</h2>
−
         <p>After collecting data, we constructed a three-dimensional model of tubulin polymerization, in which the independent variables were temperature, concentration, and energy GTP.</p>
+
         <p>We established models using statistical mechanics describing the process of tubulin polymerization in vitro.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <div class="note-content">
 
         <div class="note-content">
 
         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
−
         <p>Sent recombined plasmids including pYD1-flic-mGFP、pYCα-PETase、pYCα- XynA、pYCα- BG、pYCα- EG and pYCα-CBH sequencing.</p>
+
         <p>Sent recombined plasmids including pYDF-eGFP、pYCα-FliC(PETase)、pYCα-FliC(XynA)、pYCα-FliC(BG)、pYCα-FliC(EG) and pYCα-FliC(CBH) sequencing.</p>
 
         <p>We succeeded in electronic transformation way, transforming pYD1-mCherry into yeast competent cells and screening on SD plate (added Leu).  </p>
 
         <p>We succeeded in electronic transformation way, transforming pYD1-mCherry into yeast competent cells and screening on SD plate (added Leu).  </p>
 +
        <h2>Dry Lab</h2>
 +
        <p>Built a mathematical model to show the process of polymerization for flagellins in vitro.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
 
         <p>We transformed pYD1-mCherry into yeast competent cells EBY100 in electronic transformation way and screened on SD plate (added Leu), expanding culture.</p>
 
         <p>We transformed pYD1-mCherry into yeast competent cells EBY100 in electronic transformation way and screened on SD plate (added Leu), expanding culture.</p>
−
         <p>Plasmids ( pYCα-PETase、pYCα- XynA、pYCα- BG、pYCα- EG、pYCα-CBH )had successful sequenced.</p>
+
         <p>Plasmids ( pYCα-FliC(PETase)、pYCα-FliC(XynA)、pYCα-FliC(BG)、pYCα-FliC(EG)、pYCα-FliC(CBH))had successful sequenced.</p>
−
         <p>Reconstructed pYD1-flic-Mgfp.</p>
+
         <p>Reconstructed pYDF-eGFP.</p>
 
         <h2>Dry Lab</h2>
 
         <h2>Dry Lab</h2>
−
         <p>We analyzed the structure of tubulin and flagellin by computer.</p>
+
         <p>After collecting data, we constructed a three-dimensional model of tubulin polymerization, in which the independent variables were temperature, concentration, and energy GTP.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <div class="note-content">
 
         <div class="note-content">
 
         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
−
         <p>We transformed pYD1-α、pYD1-β into yeast competent cells EBY100 successfully.</p>
+
         <p>We transformed pYD1-α-tubulin、pYD1-β-tubulin into yeast competent cells EBY100 successfully.</p>
−
         <p>We constructed pYD1-flic-mGFP successfully.</p>
+
         <p>We constructed pYDF-eGFP successfully.</p>
−
         <p>We transformed pYD1-flic-mGFP into yeast competent cells EBY100 in electronic transformation way and screened on SD plate (added leu), expanding culture.</p>
+
         <p>We transformed pYDF-eGFP into yeast competent cells EBY100 in electronic transformation way and screened on SD plate (added leu), expanding culture.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <div class="note-content">
 
         <div class="note-content">
 
         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
−
         <p>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.</p>
+
         <p>We transformed pYCα-α-tubulin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-β-tubulin-mGFP、pYCα-mCherry (positive) into yeast competent cells INVSc1 that was cultured on SD plates including leucine, tryptophan, histidine to screen.</p>
−
         <p>A strong reducing agent DDT was 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.</p>
+
         <p>A strong reducing agent DDT was 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 pYDF-eGFP. 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.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <div class="note-content">
 
         <div class="note-content">
 
         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
−
         <p>We successfully transformed plasmids(pYCα-α、pYCα-β、pYCα- mCherry-α、pYCα-mCherry) into invsc1.</p>
+
         <p>We successfully transformed plasmids(pYCα-α-tubulin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-mCherry) into INVSc1.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <div class="note-content">
 
         <div class="note-content">
 
         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
−
         <p>Saccharomyces cerevisiae that was transformed in plasmids(pYCα-α、pYCα-β、pYCα- mCherry-α、pYCα-mCherry) successfully expressed the target protein.</p>
+
         <p>Saccharomyces cerevisiae that was transformed in plasmids(pYCα-α-tubulin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-mCherry) successfully expressed the target protein.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <div class="note-content">
 
         <div class="note-content">
 
         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
−
         <p>Through coomassie blue staining, target proteins expressed by pYD1-α、pYD1-β were successfully tested . Western Blot was used in chemical coloration to confirm the target protein.</p>
+
         <p>Through coomassie blue staining, target proteins expressed by pYD1-α-tubulin、pYD1-β-tubulin were successfully tested . Western Blot was used in chemical coloration to confirm the target protein.</p>
 
         <p>We constructed models to simulate the actual working state of Tubulin and Flagellum.</p>
 
         <p>We constructed models to simulate the actual working state of Tubulin and Flagellum.</p>
 
         </div>
 
         </div>
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         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
 
         <p>The effect of substrate DAB staining on protein detection was not ideal in Western blot. We attempted to use a more sensitive ECL color-developing method and achieved successfully.</p>
 
         <p>The effect of substrate DAB staining on protein detection was not ideal in Western blot. We attempted to use a more sensitive ECL color-developing method and achieved successfully.</p>
−
         <p>Saccharomyces cerevisiae that was transformed in plasmids(pYD1-α、pYD1-β) successfully expressed the target protein in cells.</p>
+
         <p>Saccharomyces cerevisiae that was transformed in plasmids(pYD1-α-tubulin、pYD1-β-tubulin) successfully expressed the target protein in cells.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <div class="note-content">
 
         <div class="note-content">
 
         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
−
         <p>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.</p>
+
         <p>We transformed pYCα-α-tubulin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-β-tubulin-mGFP、pYCα-mCherry (positive) into yeast competent cells INVSc1 that was cultured on SD plates including leucine, tryptophan, histidine to screen.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <div class="note-content">
 
         <div class="note-content">
 
         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
−
         <p>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.</p>
+
         <p>Through coomassie blue staining, target proteins expressed by pYCα-α-tubuin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-β-tubulin-mGFP、pYCα-mCherry (positive) were successfully tested . Western Blot in ECL color-developing method confirmed the target protein.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <div class="note-content">
 
         <div class="note-content">
 
         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
−
         <p>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.</p>
+
         <p>Through coomassie blue staining, target proteins expressed by pYCα-FliC(PETase)、pYCα-FliC(XynA)、pYCα-FliC(BG)、pYCα-FliC(EG)、pYCα-FliC(CBH) were successfully tested . Western Blot in ECL color-developing method confirmed the target protein.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
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         <div class="note-content">
 
         <div class="note-content">
 
         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
−
         <p>Through coomassie blue staining, target proteins expressed by pYCα-FliC- BG、pYCα- FliC-EG、pYCα-FliC-CBH were successfully tested . Western Blot in ECL color-developing method confirmed the target protein.</p>
+
         <p>Through coomassie blue staining, target proteins expressed by pYCα-FliC(BG)、pYCα- FliC(EG)、pYCα-FliC(CBH) were successfully tested . Western Blot in ECL color-developing method confirmed the target protein.</p>
 
         <h2>Dry Lab</h2>
 
         <h2>Dry Lab</h2>
 
         <p>The effect of tubulin concentration and GTP concentration on the length and intensity of microtubule polymerization was analyzed by establishing ordinary differential equations.</p>
 
         <p>The effect of tubulin concentration and GTP concentration on the length and intensity of microtubule polymerization was analyzed by establishing ordinary differential equations.</p>
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         <div class="note-content">
 
         <div class="note-content">
 
         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
−
         <p>Through coomassie blue staining, target proteins expressed by pYCα-FliC-PETase、pYCα-FliC (XynA) were successfully tested . Western Blot in ECL color-developing method confirmed the target protein.</p>
+
         <p>Through coomassie blue staining, target proteins expressed by pYCα-FliC(PETase)、pYCα-FliC(XynA) were successfully tested . Western Blot in ECL color-developing method confirmed the target protein.</p>
 
         </div>
 
         </div>
 
         </div>
 
         </div>
Line 410: Line 417:
 
         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
 
         <p>Make the standard curve of XynA enzyme activity.<br />
 
         <p>Make the standard curve of XynA enzyme activity.<br />
−
The activity of XynA enzyme expressed by pYCα-FliC -XynA was successfully tested.<br />
+
The activity of XynA enzyme expressed by pYCα-FliC(XynA) was successfully tested.<br />
 
Purified proteins for polymerization in vitro.</p>
 
Purified proteins for polymerization in vitro.</p>
 
<h2>Dry Lab</h2>
 
<h2>Dry Lab</h2>
Line 422: Line 429:
 
         <div class="note-content">
 
         <div class="note-content">
 
         <h2>Wet Lab</h2>
 
         <h2>Wet Lab</h2>
−
         <p>The activity of XynA enzyme expressed by pYD1-FliC -XynA was successfully tested.<br />
+
         <p>The activity of XynA enzyme expressed by pYD1-FliC(XynA) was successfully tested.<br />
 
Western Blot in ECL color-developing method confirmed the target protein (expressed by pYCα-FliC (XynA) and pYCα-mcherry) purified from supernatant.<br />
 
Western Blot in ECL color-developing method confirmed the target protein (expressed by pYCα-FliC (XynA) and pYCα-mcherry) purified from supernatant.<br />
−
Attempt to polymerize microtubule and flagellar filaments in vitro.</p>
+
Attempt to polymerize microtubule and flagellar filaments in vitro.<br/>
 +
The polymerized microtubules on yeast surface were observed with High Resolution Transmission Electron Microscopy (HRTEM).
 +
</p>
 
<h2>Dry Lab</h2>
 
<h2>Dry Lab</h2>
 
<p>The relationship between flagellin polymerization and time was used to guide the culture time of yeast.</p>
 
<p>The relationship between flagellin polymerization and time was used to guide the culture time of yeast.</p>

Latest revision as of 02:24, 2 November 2017

BNU-China

Sorry, the image is not spupported by your browser.Week 1 (March.19 - March.25)

Wet Lab

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

Sorry, the image is not spupported by your browser.Week 2 (March.26 - April.01)

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.

Sorry, the image is not spupported by your browser.Week 3 (April.02 - April.08)

Wet Lab

We had a brainstorm about yeast surface display.

Sorry, the image is not spupported by your browser.Week 4 (April.09 - April.15)

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.

Sorry, the image is not spupported by your browser.Week 5 (April.16 - April.22)

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.

Sorry, the image is not spupported by your browser.Week 6 (April.23 - April.29)

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 could 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 could finally display the microtubule and flagellum on the cell surface. And then, we could use this upgraded system to facilitate small molecules screening.

Dry Lab

Built a mathematical model to test the theoretical possibility of polymerization for tubulins and flagellins in vitro.

Sorry, the image is not spupported by your browser.Week 7 (April.30 - May.06)

Wet Lab

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

Searched DNA sequences (FliC、PETase、XynA、BG、EG、CBH)

Dry Lab

Tried to find the most appropriate length for linker to improve the possibility of polymerization.

Sorry, the image is not spupported by your browser.Week 8 (May.07 - May.13)

Wet Lab

Obtained 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.

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

Dry Lab

We studied the mechanisms of tubulin and flagellin polymerization.

Sorry, the image is not spupported by your browser.Week 9 (May.14 - May.20)

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 used agarose gel electrophoresis to prove true. Then, we utilized infusion technology to ligate target genes with pYD1 or pYCα，and had an agarose gel electrophoresis to prove true. Moreover, we transformed vectors we constructing into competent Escherichia coli cells(DH5α). Screened on LB plate(added Ampicillin). Sent to the company to make sure the sequence.

Dry Lab

We discussed the main modeling problem in our project.

Sorry, the image is not spupported by your browser.Week 10 (May.21 - May.27)

Wet Lab

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

Dry Lab

We learned as many as possible modeling which maybe useful for our work.

Sorry, the image is not spupported by your browser.Week 11 (May.28 - June.03)

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.

Continued constructing plasmids.

Dry Lab

We learned as many as possible modeling which maybe useful for our work.

Sorry, the image is not spupported by your browser.Week 12 (June.04 - June.10)

Wet Lab

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

Continued transforming recombined plasmids in Saccharomyces cerevisiae (EBY100 & IVSCc1) competent cells using chemical transforming method.

Dry Lab

We discussed to make sure what problem we were about to solve.

Sorry, the image is not spupported by your browser.Week 13-15 (June.11 - July.01)

Wet Lab

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

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

Dry Lab

We discussed to make sure what method we were about to use and what model we were about to build.

Sorry, the image is not spupported by your browser.Week 16 (July.02 - July.08)

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-FliC(PETase)、pYCα-FliC(XynA)、pYCα-FliC(BG)、pYCα-FliC(EG)、pYCα-FliC(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 could express secretory proteins. Screened strains on LB plate (added Ampicillin). Sent sequencing.

Recombined plasmid pYD1-FliC-Mgfp was constructed in the same way with pYCα-FliC(PETase)、pYCα-FliC(XynA)、pYCα-FliC(BG)、pYCα-FliC(EG)、pYCα-FliC(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

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

Sorry, the image is not spupported by your browser.Week 17 (July.09 - July.15)

Wet Lab

Sent recombined plasmids including pYDF-eGFP、pYCα-FliC(PETase)、pYCα-FliC(XynA)、pYCα-FliC(BG)、pYCα-FliC(EG) and pYCα-FliC(CBH) sequencing.

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

Dry Lab

Built a mathematical model to show the process of polymerization for flagellins in vitro.

Sorry, the image is not spupported by your browser.Week 18 (July.16 - July.22)

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-FliC(PETase)、pYCα-FliC(XynA)、pYCα-FliC(BG)、pYCα-FliC(EG)、pYCα-FliC(CBH))had successful sequenced.

Reconstructed pYDF-eGFP.

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.

Sorry, the image is not spupported by your browser.Week 19 (July.23 - July.29)

Wet Lab

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

We constructed pYDF-eGFP successfully.

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

Sorry, the image is not spupported by your browser.Week 20 (July.23 - July.29)

Wet Lab

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

Sorry, the image is not spupported by your browser.Week 21 (July.30 - August.05)

Wet Lab

We transformed pYCα-α-tubulin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-β-tubulin-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 was 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 pYDF-eGFP. 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.

Sorry, the image is not spupported by your browser.Week 22 (August.06 - August.12)

Wet Lab

We successfully transformed plasmids(pYCα-α-tubulin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-mCherry) into INVSc1.

Sorry, the image is not spupported by your browser.Week 23 (August.13 - August.19)

Wet Lab

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

Sorry, the image is not spupported by your browser.Week 24 (August.20 - August.26)

Wet Lab

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

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

Sorry, the image is not spupported by your browser.Week 25 (August.12 – September.02 )

Wet Lab

The effect of substrate DAB staining on protein detection was 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-tubulin、pYD1-β-tubulin) successfully expressed the target protein in cells.

Sorry, the image is not spupported by your browser.Week 26 (September.03 - September.09)

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

Sorry, the image is not spupported by your browser.Week 27 (September.10 - September.16)

Wet Lab

We transformed pYCα-α-tubulin、pYCα-β-tubulin、pYCα- mCherry-α-tubulin、pYCα-β-tubulin-mGFP、pYCα-mCherry (positive) into yeast competent cells INVSc1 that was cultured on SD plates including leucine, tryptophan, histidine to screen.

Sorry, the image is not spupported by your browser.Week 28 (September.17 - September.23)

Wet Lab

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

Sorry, the image is not spupported by your browser.Week 29 (September.24 - September.30)

Wet Lab

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

Sorry, the image is not spupported by your browser. Week 30 (October.1 - October.7)

Wet Lab

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

Dry Lab

The effect of tubulin concentration and GTP concentration on the length and intensity of microtubule polymerization was analyzed by establishing ordinary differential equations.

Sorry, the image is not spupported by your browser. Week 31 (October.8 - October.15)

Wet Lab

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

Sorry, the image is not spupported by your browser. Week 32 (October.16 - October.23)

Wet Lab

Make the standard curve of XynA enzyme activity.
The activity of XynA enzyme expressed by pYCα-FliC(XynA) was successfully tested.
Purified proteins for polymerization in vitro.

Dry Lab

Simulation of the efficiency of flagellin aggregation on yeast surface by flagellin concentration and yeast concentration.

Sorry, the image is not spupported by your browser. Week 33 (October.24 - November.1)

Wet Lab

The activity of XynA enzyme expressed by pYD1-FliC(XynA) was successfully tested.
Western Blot in ECL color-developing method confirmed the target protein (expressed by pYCα-FliC (XynA) and pYCα-mcherry) purified from supernatant.
Attempt to polymerize microtubule and flagellar filaments in vitro.
The polymerized microtubules on yeast surface were observed with High Resolution Transmission Electron Microscopy (HRTEM).

Dry Lab

The relationship between flagellin polymerization and time was used to guide the culture time of yeast.

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