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<link type="text/css" rel="stylesheet" href="https://2017.igem.org/Template:BNU-China/CSS/final?action=raw&ctype=text/css" /> | <link type="text/css" rel="stylesheet" href="https://2017.igem.org/Template:BNU-China/CSS/final?action=raw&ctype=text/css" /> | ||
+ | <script src="jquery-3.1.1.min.js"></script> | ||
<script type="text/javascript" src="https://2017.igem.org/Template:BNU-China/Javascript/final?action=raw&ctype=text/javascript"></script> | <script type="text/javascript" src="https://2017.igem.org/Template:BNU-China/Javascript/final?action=raw&ctype=text/javascript"></script> | ||
<script type="text/javascript" src="https://2017.igem.org/Template:BNU-China/Javascript/index?action=raw&ctype=text/javascript"></script> | <script type="text/javascript" src="https://2017.igem.org/Template:BNU-China/Javascript/index?action=raw&ctype=text/javascript"></script> | ||
− | + | <style type="text/css"> | |
#content { | #content { | ||
background-color: rgb(247,237,183); | background-color: rgb(247,237,183); | ||
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font-size: 35px; | font-size: 35px; | ||
font-weight: bold; | font-weight: bold; | ||
+ | display: inline-block; | ||
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} | } | ||
.week h2{ | .week h2{ | ||
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@media screen and (max-width:911px){ | @media screen and (max-width:911px){ | ||
+ | .week{ | ||
+ | position: relative; | ||
+ | left: 10%; | ||
+ | width: 78%; | ||
+ | } | ||
.week p{ | .week p{ | ||
font-size: 14px; | font-size: 14px; | ||
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} | } | ||
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<div class="week"> | <div class="week"> | ||
− | <h1>Week 1 (March.19 - March.25)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;"><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 1 (March.19 - March.25)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
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− | <h1>Week 2 (March.26 - April.01)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 2 (March.26 - April.01)</h1> |
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− | <h1>Week 3 (April.02 - April.08)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 3 (April.02 - April.08)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
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</div> | </div> | ||
<div class="week"> | <div class="week"> | ||
− | <h1>Week 4 (April.09 - April.15)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 4 (April.09 - April.15)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
<h2>Wet Lab</h2> | <h2>Wet Lab</h2> | ||
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</div> | </div> | ||
<div class="week"> | <div class="week"> | ||
− | <h1>Week 5 (April.16 - April.22)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 5 (April.16 - April.22)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
<h2>Wet Lab</h2> | <h2>Wet Lab</h2> | ||
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<div class="week"> | <div class="week"> | ||
− | <h1>Week 6 (April.23 - April.29)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 6 (April.23 - April.29)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
<h2>Wet Lab</h2> | <h2>Wet Lab</h2> | ||
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</div> | </div> | ||
<div class="week"> | <div class="week"> | ||
− | <h1>Week 7 (April.30 - May.06)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 7 (April.30 - May.06)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
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<div class="week"> | <div class="week"> | ||
− | <h1>Week 8 (May.07 - May.13)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 8 (May.07 - May.13)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
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<div class="week"> | <div class="week"> | ||
− | <h1>Week 9 (May.14 - May.20)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 9 (May.14 - May.20)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
<h2>Wet Lab</h2> | <h2>Wet Lab</h2> | ||
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</div> | </div> | ||
<div class="week"> | <div class="week"> | ||
− | <h1>Week 10 (May.21 - May.27)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 10 (May.21 - May.27)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
<h2>Wet Lab</h2> | <h2>Wet Lab</h2> | ||
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</div> | </div> | ||
<div class="week"> | <div class="week"> | ||
− | <h1>Week 11 (May.28 - June.03)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 11 (May.28 - June.03)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
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<div class="week"> | <div class="week"> | ||
− | <h1>Week 12 (June.04 - June.10)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 12 (June.04 - June.10)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
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<div class="week"> | <div class="week"> | ||
− | <h1>Week 13-15 (June.11 - July.01)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 13-15 (June.11 - July.01)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
<h2>Wet Lab</h2> | <h2>Wet Lab</h2> | ||
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<div class="week"> | <div class="week"> | ||
− | <h1>Week 16 (July.02 - July.08)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 16 (July.02 - July.08)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
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− | <h1>Week 17 (July.09 - July.15)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 17 (July.09 - July.15)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
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− | <h1>Week 18 (July.16 - July.22)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 18 (July.16 - July.22)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
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− | <h1>Week 19 (July.23 - July.29)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 19 (July.23 - July.29)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
<h2>Wet Lab</h2> | <h2>Wet Lab</h2> | ||
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− | <h1>Week 20 (July.23 - July.29)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 20 (July.23 - July.29)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
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− | <h1>Week 21 (July.30 - August.05)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 21 (July.30 - August.05)</h1> |
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− | <h1>Week 22 (August.06 - August.12)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 22 (August.06 - August.12)</h1> |
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− | <h1>Week 23 (August.13 - August.19)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 23 (August.13 - August.19)</h1> |
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− | <h1>Week 24 (August.20 - August.26)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 24 (August.20 - August.26)</h1> |
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− | <h1>Week 25 (August.12 – September.02 )</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 25 (August.12 – September.02 )</h1> |
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− | <h1>Week 26 (September.03 - September.09)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 26 (September.03 - September.09)</h1> |
<div class="note-content"> | <div class="note-content"> | ||
<!-- <h2>Wet Lab</h2> --> | <!-- <h2>Wet Lab</h2> --> | ||
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− | <h1>Week 27 (September.10 - September.16)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 27 (September.10 - September.16)</h1> |
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− | <h1>Week 28 (September.17 - September.23)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 28 (September.17 - September.23)</h1> |
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− | <h1>Week 29 (September.24 - September.30)</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">Week 29 (September.24 - September.30)</h1> |
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− | <h1>10.1</h1> | + | <h1><img src="https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png" alt="Sorry, the image is not spupported by your browser." style="display:inline-block;width:10%;">10.1</h1> |
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<!-- <h2>Wet Lab</h2> --> | <!-- <h2>Wet Lab</h2> --> |
Revision as of 13:29, 7 October 2017
![Sorry, the image is not spupported by your browser.](https://static.igem.org/mediawiki/2017/b/ba/T--BNU-China--notebookLogo.png)
Week 1 (March.19 - March.25)
Wet Lab
Read excellent literatures about synthetic biology and projects on iGEM Official Website.
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.
Week 3 (April.02 - April.08)
Wet Lab
We had a brainstorm about yeast surface display.
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.
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.
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 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.
Week 7 (April.30 - May.06)
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.
Week 8 (May.07 - May.13)
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.
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 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.
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、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.
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.
Continue constructing plasmids.
Week 12 (June.04 - June.10)
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.
Week 13-15 (June.11 - July.01)
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.
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-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.
Week 17 (July.09 - July.15)
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).
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-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.
Week 19 (July.23 - July.29)
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.
Week 20 (July.23 - July.29)
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.
Week 21 (July.30 - August.05)
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.
Week 22 (August.06 - August.12)
Wet Lab
We successfully transformed plasmids(pYCα-α、pYCα-β、pYCα- mCherry-α、pYCα-mCherry) into invsc1.
Week 23 (August.13 - August.19)
Wet Lab
Saccharomyces cerevisiae that was transformed in plasmids(pYCα-α、pYCα-β、pYCα- mCherry-α、pYCα-mCherry) successfully expressed the target protein.
Week 24 (August.20 - August.26)
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.
Week 25 (August.12 – September.02 )
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.
Week 26 (September.03 - September.09)
Western Blot in ECL color-developing method confirmed the target protein was expressed.
Week 27 (September.10 - September.16)
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.
Week 28 (September.17 - September.23)
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.
Week 29 (September.24 - September.30)
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.
10.1
Protein purification
Plasmids pYC, -PETase, pYC, alpha - XynA, pYC - - BG, pYC - - EG and pYC - -CBH were successfully transformed into Saccharomyces cerevisiae invsc1.
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