Team:USTC-China/groupmeetings

From 2012.igem.org

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     <div class="col_list">
     <div class="col_list">
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         <h2>Weeks</h2>
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         <h2>Date</h2>
<ul>
<ul>
-
    <li id="yhl">Jun 28-July 2</li>
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    <li id="wyc1">18th,Feb</li>
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    <li id="swl">July 3-July 9</li>
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    <li id="xwc">25th,Feb</li>
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    <li id="zlc">July 10-July 16</li>
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    <li id="xc">3rd, Mar</li>
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    <li id="fzz">July 17-July 23</li>
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    <li id="zxc">10th,Mar</li>
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    <li id="shl">July 24-July 30</li>
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    <li id="tf">17th,Mar</li>
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    <li id="ml">July 31-Aug 6</li>
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    <li id="wyc2">24th,Mar</li>
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    <li id="mps">Aug 7-Aug 13</li>
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    <li id="xyg1">7th, Apr</li>
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             <li id="yfl">Aug 14-Aug 20</li>
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             <li id="xyg2">14th,Apr</li>
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    <li id="dz">Aug 21-Aug 27</li>
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    <li id="wyc3">6th, May</li>
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    <li id="lna">Aug 28-Sep 3</li>
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    <li id="xzl">13th,May</li>
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    <li id="jhp">Sep 4-Sep 10</li>
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    <li id="jwh">21st,May</li>  
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    <li id="xlz">Sep 11-Sep 17</li>
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-
            <li id="wyy">Sep 18-Sep 24</il>
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    <li id="ytl">Sep 25-Oct 1</li>
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            <li id="nw">Oct 2-Oct 8</li>
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            <li id="yz">Oct 9-Oct 15</il>  
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</ul>
</ul>
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<div id="col_left">
<div id="col_left">
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<div class="bio" id="yhlbio">
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<div class="bio" id="wyc1bio">
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<h1>Jun 28-July 2</h1>
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<h1>18th, Feb</h1>
-
2011.6.28
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<p>Place: Room 363, Life Science building
</p>
</p>
-
<hr />
+
<p>Instructor: Mr.Hong
-
<p>cultivate the bacteria of <a href="http://partsregistry.org/Part:BBa_K557000" class="external text" rel="nofollow">cheZ</a> deficiency(RP1616),and the Control group(RP437).
+
-
check the resistibility of RP1616 and RP437
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-
result:both of the two groups are of none resistibility.
+
-
members:Siwei Luo, Fangzhou Zhao, Zhilin Chen...
+
-
</p><p>2011.6.29
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</p>
</p>
-
<hr />
+
<p>Recordist: Wuyang Chen
-
<p>check the motility of RP1616 strain and RP437 strain
+
-
cultivate Top10 strain
+
-
result: the bacterial plaque of RP1616 is smaller than that of RP437, implying that RP1616 is of <a href="http://partsregistry.org/Part:BBa_K557000" class="external text" rel="nofollow">cheZ</a> deficiency, causing the decreased motility of the bacteria.
+
-
</p><p><br />
+
-
6.30
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</p>
</p>
<hr />
<hr />
-
<p>prepare for the competent cell of Top10 strain cultivated on 6.29
+
<p>I. Discuss about our project:<br>
-
extract the genome of Top10 strain and use it as complates to run PCR of CheZ
+
1. SNS:<br>
-
result: the concentration of PCR result is too low to continue the experiments.
+
To complete this project is hardly possible. While it is likely to realize part of it, such as a system which is able to create and sense signals. But we need to confer our project some new meanings.<br>
-
</p><p>7.1
+
1) About making a signal disappear, according to Mr. Hong, we can add a rapid degradation label onto the signal.<br>
 +
2) We are afraid that the colony is too small to create an appropriate concentration gradient. According to Mr. Hong, it won’t be a big problem.<br>
 +
<br>
 +
2. Solve the graph colouring problem using E.coli<br>
 +
1) In fact, we will only use E.coli to emit light in different colors, and all the algorithm for solving graph coloring problem is processed artificially.<br>
 +
2) We need to consult professors in Chemistry Department about constructing structures using ssDNA.<br>
 +
3) The scale of DNA strand is far too small than that of E.coli. <br>
 +
4) The fusion protein of Zn finger and the Ag43 would be very useful. We can think about how to make use of it.<br>
 +
<br>
 +
3. Fatigue of specific stimulus<br>
 +
1) comK is a protein produced by bacillus subtilis and “competence” is a state of it. The possibility of turning into competence state is proportional to the amount of comK. We can substitute the protein involved in competence state for the protein we need.<br>
 +
2) The system can be used as a special lock.<br>
 +
3) How to make circuit one recover its function as quick as possible?<br>
 +
a) by means of protease?<br>
 +
b) by means of aptamer?<br>
 +
<br>
 +
II. Set up groups to continue brainstorm<br>
 +
<br>
 +
III. Make plans for the whole term.<br>
</p>
</p>
-
<hr />
+
 
-
<p>conduct PCR of CheZ again
+
-
ligate the CheZ DNA segment to plasmid PSb1C3, and transform it into Top10 competent cells
+
-
result:as the picture shows.
+
-
</p><p>7.2
+
-
</p>
+
-
<hr />
+
-
<p>pick up single colony from the Petri dish to reproduce, in which the competent cells are cultivated and use them colony PCR
+
-
result:The bacteria grow up well on dishes with chloromycetin resistency，verifying that the transformation is successfull
+
-
while the concentration of PCR product is pretty high,demonstrating that the plasmids indeed contains the sagment CheZ.
+
-
</p><p><br />
+
<div class="clear"></div>
<div class="clear"></div>
 +
</div>
</div>
-
<div class="bio" id="swlbio">
+
<div class="bio" id="xwcbio">
-
<h1>July 3-July 9</h1>
+
<h1>25th, Feb</h1>
-
 
+
<p>Place: Room 363, Life Science building
-
7.3
+
</p>
</p>
-
<hr />
+
<p>Instructor: Mr.Hong, Yinghong Lan
-
<p>extract the plasmids from the reproduced bacteria and send it to check the base sequence
+
-
result: the concentration of the plasmids extracted is about 500ng/uL
+
-
</p><p>7.4
+
</p>
</p>
-
<hr />
+
<p>Recordist: Xingwen Chen
-
<p>extract the standard parts LuxPR and Terminator from plate sent by iGEM registry 2010 and put them into Top10 strain by transformation
+
-
(LuxPr: Plate 2,24C  Terminator&nbsp;: Plate 1,6O)
+
-
result: the bacteria on the plate with Amp resistency grows well
+
-
7.5
+
</p>
</p>
<hr />
<hr />
-
<p>pick up single colony from the Petri dish to reproduce in the liquid culture medium
+
<p>Continue brainstorming.<br>
-
</p><p><br />
+
I.Three ideas we have put forward before:<br>
-
7.6
+
  1.Bacterio-SNS (Social Networking Services )<br>
 +
    problem1.The signal molecules may exist for too long.<br>
 +
    solution：Add a degradation tag to the signal protein for the cell to degrade it quickly.<br>
 +
    problem2.How to construct a memory system?<br>
 +
    There are several ways but which is the most suitable?
 +
    problem3.Does CDK exist in bacteria? <br>
 +
    We can't get it until we find a paper about that.<br>
 +
    problem4.We should consider what kinds of result we will achieve.<br><br>
 +
  2.Solve graph coloring problem with DNA and visualize the result with E.coli.<br>
 +
    problem1.How to construct the complex DNA structure?  <br>
 +
    We can ask a professor who are studying this.<br>
 +
    problem2.Bacteria are much bigger than DNA,thus the visualization may be impossible.<br><br>
 +
  3.E.coli mimic specific stimulation fatigue <br>
 +
    problem1.The idea will actually be quite boring if our purpose is only to mimic.<br>
 +
    Think of something new which can apply the system.<br>
 +
    problem2.The signal molecules may exist for too long and signals may influence each other.<br>
 +
    Change the signal and consider some physical signals such as temperature and light.<br><br><br>
 +
II.New Ideas<br>
 +
  Zhou Shan says:<br>
 +
  1.Bacteriaphage therapy<br><br>
 +
  2.Synthetic biology moving into the clinic<br><br>
 +
  3.improve bacteria tolerance：<br>
 +
    a)efflux pump<br>
 +
    b)heat shock proteins<br>
 +
    c)membrane modification<br>
 +
    d)general stress respond<br>
 +
These are mostly achieved by scientists.It is wrong to repeat others unless we can come up with something new.<br><br>
 +
4.Some thoughts about nucleic acid aptamer<br>
 +
    a) Aptamer itself can be substrate.<br>
 +
    b) Two aptamers cna be combined on a single bead.<br><br><br>
 +
III.Professor Liu suggest we could do something on the basis of our work of iGEM 2011.<br>
 +
Luo Siwei, a team member of iGEM2011,says that if we must do as this,the only thing we can do is to make it more specific,which seems a little boring.And he indicates that the work last year is too complicated and lacking in aesthetics.<br><br><br>
 +
IV.About aptamer<br>
 +
We have the technology and we can start the SELEX now.But we have to work out a good application of it.<br><br><br>
 +
V.Tasks of the next two weeks<br>
 +
  Direction 1.Come up with a new idea on the basis of the work last year. <br>
 +
    a)What can we improve?<br>
 +
    b)Think about uncompleted work.<br>
 +
    c)Try to apply the signal and response system we came up with in the SNS subject.<br>
 +
d)Try to apply the circuits in the specific stimulation fatigue subject.<br>
 +
e)Read more papers,view previous works and get some inspirations.<br><br>
 +
  Direction 2.Bacterio-SNS:<br>
 +
    a) Learn more about toggle switch.<br>
 +
    b) Find out if there is something similar to CDK complex in E.coli or B.subtilis.<br>
 +
    c) Think about what effect shall we get finally.<br>
 +
  Direction 3:E.coli mimic specific stimulation fatigue:<br>
 +
    Think of an application of it.<br>
 +
 
</p>
</p>
-
<hr />
+
</div>
-
<p>extract plasmids containing LuxPR and Terminator and make enzyme digestion with EcoR1 and Spe1 in order to validate the existence of the two parts
+
 
-
recieve part Toggle Switch and rbs-Ci-ter from PKU and conduct transformation
+
<div class="bio" id="xcbio">
-
</p><p>result: Due to the short length of LuxPR and Terminator, the result of the enzyme digestion is hard to examine by electrophoresis.
+
<h1>3rd,Mar</h1>
-
</p><p>7.7
+
<p>Place: Room 363, Life Science building
</p>
</p>
-
<hr />
+
<p>Instructor: Mr.Hong
-
<p>result: there are a few colonies with part Toggle switch grown while no colony of rbs-Ci-ter appeared.
+
-
</p><p>7.8
+
</p>
</p>
-
<hr />
+
<p>Recordist: Xi Chen
-
<p>pick up single colony with part Toggle switch .
+
-
extract the standard parts LuxPR and Terminator from plate sent by iGEM registry 2011 and put them into Top10 strain by transformation
+
-
</p><p>7.9
+
</p>
</p>
<hr />
<hr />
-
<p>pick up single colony with LuxPR &amp; terminator.
+
<p>
-
extract plasmids containing toggle switch to conduct PCR.
+
I.journal club<br>
-
</p><p>process PCR with plasmids aptamer-CheZ
+
Chen Wuyang, who talked firstly, introduced a kind of marine photosynthetic bacterium Rhodulum sulfidophilum which can produces extracellular nucleic acids. These were found to be produced concomitantly with cell growth. However, the mechanism of this phenomenon had not been figured out, instead, someone hypothesised that it is the process of lysis of a subfraction of cells that makes this happen. After that, the speaker addressed that using engineered plasmids and the bacterium can make the production of extracellular nucleic acid under control which had been proved by anther paper.<br><br>
-
result:as the picture shows.
+
 
-
the base sequence of CheZ extracted before is proved right.
+
After that Zheng Xuexuan gave us a presentation about nanorobot. This autonomous DNA nanorobot was controlled by an aptamer-encoded logic gate, enabling it show different function in different cues. What’s more this kind of device can be loaded with a variety of materials in a highly organized fashion.<br><br><br>
-
</p><p><br />
+
 
-
</p><p>
+
II.Introduction of SELEX<br>
-
<div class="clear"></div>
+
We invited Chen Liang, a graduate student, to refer us some knowledge about SELEX( Systematic Evolution of Ligands by Exponential Enrichment).In sum, he talked about the definition about this technology and the main round of this method. Additionally, some examples also were taken so that we can comprehend in a deeper degree. <br><br>
-
</div>
+
-
<div class="bio" id="zlcbio">
+
III.Discussion about the probability of the SNS the program someone raised in the previous meeting and proposed more questions remaining fixed. 
-
<h1>July 10-July 16</h1>
+
-
7.10
 
</p>
</p>
-
<hr />
+
 
-
<p>ligate aptamer-CheZ with PSB1C3 plasmids
+
</div>
-
</p><p>extract plasmids containing LuxPR and terminator
+
 
-
result:the concentractions of plasmids LuxPR and terminator are both 600ng/ul
+
<div class="bio" id="zxcbio">
-
</p><p>7.11
+
<h1>10th,Mar</h1>
 +
<p>Place: Room 363, Life Science building
</p>
</p>
-
<hr />
+
<p>Instructor: Mr.Hong, Yinghong Lan
-
<p>transform PSB1C3 containing aptamer-CheZ into bacteria and cultivate.
+
-
7.12
+
</p>
</p>
-
<hr />
+
<p>Recordist: Zhaoxiong Chen
-
<p>carry out Double digestion of toggle switch and pSB1c3 and ligate them together.
+
-
</p><p>pick up single colony from bacteria cultivated yesterday.
+
-
</p><p>result: Both the location and the brightness of the electrophoretic bands correspond with our expectation.
+
-
</p><p>7.14
+
</p>
</p>
<hr />
<hr />
-
<p>transform the part rbs-ci-ter sent from PKU again into bacteria.
+
<p>
-
check the colors of the colonies containing toggle switch.
+
I.Xuexuan Zheng：several ideas<br>
-
result: the ratio of red vs green is 8:25, verifying the function of toggle switch.
+
1.Use lactobacillus instead of E coli as our new model for genetically engineering in order to avoid the potentially harmful outcome to human so that these technology could be used in the field ofmedicine safely.<br>
-
</p><p>7.15
+
Difficulty:<br>
 +
1)source of proper strain <br>
 +
2)synthesis of standardized part <br>
 +
3)money required<br>
 +
2.Membrane RNA<br>
 +
Difficulty:RNA in membrane is unstable, easy to desquamate and be degradated<br>
 +
3.Two directions of sifting aptamers:1.theosine homolog 2.other aptamers whose properties is similar to theosine<br><br>
 +
 
 +
II.Sitao Zhang:PNA（Peptide nucleic acid）<br>
 +
Properties:<br>
 +
1.stable at high temperature  <br> 
 +
2.the binding between PNA/DNA strands is stronger than between DNA/DNA strands  3.Better recognition capability <br>
 +
4.stable in the presence of protease and DNase<br>
 +
Functions:<br>
 +
1.bind to DNA and prevent DNA from methylation<br>
 +
2.recognize DNA with specificity<br>
 +
Usage:genetic switch and sifting<br>
 +
Difficulty:<br>
 +
1.unbearable cost <br>
 +
2.can't be standardized    <br><br>
 +
 
 +
III.Xuyuan Gao:<br>
 +
1.Use the gene of Tase and attenuator regulated by tryptophan to establish a recording net.<br>
 +
2.papers about aptamers:a kind of aptamer,KMF2-1a,could bind to some special proteins in cell membrane,then it could be transported into cell by endocytosis<br><br>
 +
 
 +
 
 +
IV.Qian Li:<br>
 +
1.Optical Control:<br>
 +
1)Synthesis a special peptide,one terminal of which act as a ligand and is able to bind to cell membrane,the other terminal is a systeine whose residue,a sulfhydryl,could bind to hydrogel with the irradiation of laser.That peptide could act as a bridge to bind cells to hydrogel.<br>
 +
2)Central section of the peptide should be designed to be a special structure which could be cut off by UVL so that the cells binded to hydrugel could desquamate.<br>
 +
2.Ferritin：Ferritin is a kind of protein looks like a box,it could transport ferrum inside it in vivo.These boxes only unlock when ph is 2.0(5.0 in procaryotic cell),shut when ph reach 7.5.<br>
 +
Application：put chemical compounds in ferritin,let E coli carry them to the area where cancer cells live and growth,then unlock ferritin,release our medicines to kill cancer cells.<br><br>
 +
 
 +
 
 +
 
 +
V.Wuyang Chen:<br>
 +
a paper about how HIV works in cell:Protein Nup358 plays a important role in the process when the viral RNA is transported into nucleolus to integrate with cell DNA<br>
 +
Idea:construct several aptamers that could bind to Nup358 in order to prevent cell DNA from being integrate with cell DNA.<br>
 +
Difficulty:Nup358 is also very important in normal physiological activity,our strategy may do harm to uncontaminated cells.<br>
 +
Some sulotion:construct aptamers that could bind to the special domain of viral capsid which is used for binding to Nup358<br><br>
 +
 
 +
Four directions of our team determined：<br>
 +
1.Qian Li:New cure for cancer <br>
 +
2.Xingwen Chen:New cure for AIDS <br>
 +
3.Xuexuan Zheng:Neutralize the effect of alcoholic drinks <br>
 +
4.Xuyuan Gao:Recording net inside cell<br>
 +
 
 +
 
</p>
</p>
-
<hr />
+
</div>
-
<p>pick up the single colony from the plate yesterday and ten hours later extract the plasmids containing Ci-terminator.
+
 
-
</p><p><br />
+
<div class="bio" id="tfbio">
-
7.16
+
<h1>17th,Mar</h1>
 +
<p>Place: Room 363, Life Science building
 +
</p>
 +
<p>Instructor: Mr.Hong, Yinghong Lan
 +
</p>
 +
<p>Recordist: Ting Fu
</p>
</p>
<hr />
<hr />
-
<p>conduct PCR with the plasmids yesterday to reproduce rbs-ci-Term.
 
-
result:failure
 
-
</p><p><br />
 
-
</div>
+
<p>
 +
Progress on our four ideas:<br>
 +
I.HIV<br>
 +
Wuyang Chen: <br>
 +
1.Introduce the structure of HIV and their functions.<br>
 +
2.Introduce the process how the HIV absorb and invade the T4 cell.<br>
 +
3.Useful details from two papers: <br>
 +
"Capsid is a dominant determinant of retrovirus infectivity in nondividing cells"<br>
 +
"HIV-1 Capsid-Cyclophilin Interactions Determine Nuclear Import Pathway-Integrati"<br><br>
-
<div class="bio" id="fzzbio">
+
Discussion:<br>
-
<h1>July 17-July 23</h1>
+
1.Application of aptamer.<br>
-
7.18-7.21
+
2.Prevention of the cellular membrane conjugation.<br>
-
----
+
3.T cell conduct suicide when it feel infected.<br><br>
-
ligate the standard part Terminator to the end of toggle switch
+
-
result: we conduct a enzyme digestion with EcoR1 and Pst1 to the final ligation product and the electrophoresis result shows that the ligation is successful.
+
It is not practical for the team to deal with HIV, however, we can simplify our idea which concerns the resistance against the virus, that is, the phage. One thought is that we make the promoter of our parts the same as the promoter of the virus expression system. When the virus invades, it switch on two set of systems: one binds the phage inhibiting its reproduction, the other makes the host suicide. This project can be applied to fermentation production.<br><br><br>
-
<div class="clear"></div>
+
-
</div>
+
-
<div class="bio" id="shlbio">
+
II.Cisplatin<br>
-
<h1>July 24-July 30</h1>
+
Qian Li:<br>
-
7.22-7.29
+
Useful info from papers: a novel aptamer KMF2-1a is internalized efficiently and specifically to the endosome of target breast cancer cells.<br><br><br>
-
----
+
-
perform the following experiments: PCR of rbs-ci-ter, enzyme digestion(E,S),ligation with LuxPR(standard part from IGEM registry digested by E,X),tranformation,plasmids extraction.
+
-
result: the concentration of plasmids with ligation gene is as high as ...
+
 +
III.Relieving alcoholism and nicotine<br>
 +
Xuexuan Zhen:<br>
 +
About the alcoholism: design a positive feedback, making the Bacillus acidi-lactici heavily express components to decompose the alcohol.<br>
 +
Key: a alcohol sensor, a positive feedback, application of QS.<br>
 +
About the nicotine:  expressions of unpleasant smell or something absorbs and relieves the nicotine when nicotine is taken by people. Some bacterium lives on nicotine as carbon source or nitrogen source. The relevant genes can be used to relieve the nicotine directly.<br><br><br>
 +
IV.The gene toutine<br>
 +
Xuyuan Gao:
 +
a routine responses only when a substance is added twice.<br>
 +
a encoded routine: a routine reponses when the amino acid is added at the right order.<br><br><br>
-
<div class="clear"></div>
+
More papers:
-
</div>
+
Jinxin Wang: a therapy on cancer, antimetabolitas which inhibits the dividing phase.
 +
Cai Li: an aptamer combines with hemoglobin.
-
<div class="bio" id="mlbio">
+
</p>
-
<h1>July 31-Aug 6</h1>
+
-
7.30-8.3
+
-
----
+
-
PCR of LuxPR-rbs-ci-ter, enzyme digestion(E,S),ligation with LuxPR(standard part from IGEM registry digested by E,X),tranformation,plasmids extraction.
+
</div>
</div>
-
<div class="bio" id="mpsbio">
+
<div class="bio" id="wyc2bio">
-
<h1>Aug 7-Aug 13</h1>
+
<h1>24th,Mar</h1>
 +
<p>Place: Room 363, Life Science building
 +
</p>
 +
<p>Instructor: Mr.Hong, Yinghong Lan
 +
</p>
 +
<p>Recordist: Wuyang Chen
 +
</p>
 +
<hr />
-
<div class="clear"></div>
+
<p>
-
</div>
+
 +
About the project of virus<br><br>
 +
I. Introduction of lambda phage<br>
 +
1. Construction and maintenance of lysogenic life cycle<br>
 +
Repressor CI is the only protein produced during lysogenic life cycle. Its functions are:<br>
 +
1) combine to OL: represses the expression of the gene downstream the promoter pL.<br>
 +
2) combine to OR1: represses the expression of the gene downstream the promoter pR.<br>
 +
3) combine to OR2: activates the expression from promoter pRM and maintains its own expression.<br><br>
-
<div class="bio" id="yflbio">
+
Transform into lytic life cycle<br>
-
<h1>Aug 14-Aug 20</h1>
+
Cro is responsible for preventing the synthesis of the repressor CI and this action shuts off the possibility of establishing lysogeny. It has two effects:<br>
 +
1. It prevents the synthesis of repressor via the maintenance circuit; that is, it prevents transcription via pRM .<br>
 +
2. It also inhibits the expression of early genes from both pL and pR Cro achieves its function by binding to the same operators as (el) repressor protein.<br><br>
 +
II. Idea about our project<br>
 +
1. Promoter: same as the promoter in lambda phage.<br>
 +
2. Aaptamer: try to bind the receptor the phage uses to anchor onto the membrane of bacteria. <br>
 +
3. Genes for defence: <br>
 +
1) gene for suicide.<br>
 +
2) gene cI.<br>
 +
3) gene whose products can inhibit the expression of Cro.<br>
 +
 +
 +
</p>
</div>
</div>
-
<div class="bio" id="nwbio">
+
<div class="bio" id="xyg1bio">
-
<h1></h1>
+
<h1>7th,Apr</h1>
 +
<p>Place: Room 363, Life Science building
 +
</p>
 +
<p>Instructor: Mr.Hong
 +
</p>
 +
<p>Recordist: Xuyuan Gao
 +
</p>
 +
<hr />
 +
<p>
 +
The main component of the “Gene passwords” in this conversation:<br><br>
 +
1.Introduce the rudiments of the attenuator:<br>
 +
1)The gene structure of the attenuator;<br>
 +
2)The essence of why attenuator can reduce the production of amino acid.(Take the tryptophan attenuator for example and advance a personal opinion)<br>
 +
3)The mechanism of how the attenuator can reduce the production of amino acid.<br><br>
 +
2.Improve the “Gene passwords” :<br>
 +
Use some step of one circular biochemistry reaction as the nodes for control.<br>
-
<div class="clear"></div>
+
</p>
</div>
</div>
-
<div class="bio" id="ytlbio">
 
-
<h1></h1>
 
 +
<div class="bio" id="xyg2bio">
 +
<h1>14th,Apr</h1>
 +
<p>Place: Room 363, Life Science building
 +
</p>
 +
<p>Instructor: Mr.Hong, Yinghong Lan
 +
</p>
 +
<p>Recordist: Xuyuan Gao
 +
</p>
 +
<hr />
-
<div class="clear"></div>
+
<p>
 +
The main component of the “Gene passwords” in this conversation:<br><br>
 +
The data of the ”Gene passwords” (Attach the lecture for the “Gene passwords”)Except me, I need two other teammates for this group. For this is an original project, and there is no former production that we can borrow, and we don’t have the parts that have been made out. Though the second part of the project is really difficult, we may complete the first second part of the project, but I’m not sure that we can complete it certainly. If you are interested in the project, you can join me.<br>
 +
 
 +
</p>
</div>
</div>
-
<div class="bio" id="dzbio">
 
-
<h1></h1>
 
-
</div>
 
-
<div class="bio" id="lnabio">
+
<div class="bio" id="wyc3bio">
-
<h1></h1>
+
<h1>6th, May</h1>
 +
<p>Place: Room 363, Life Science building
 +
</p>
 +
<p>Instructor: Mr.Hong
 +
</p>
 +
<p>Recordist: Wuyang Chen
 +
</p>
 +
<hr />
-
<div class="clear"></div>
+
<p>
-
</div>
+
Project of phage<br><br>
 +
1. Part for initiating transcription: pRM, BBa_I714889<br>
 +
2. Part for inhibit the lambda from turning into lytic life cycle: antisense RNA<br>
 +
This is the core of our project, we need to deeply survey this part.<br><br>
-
<div class="bio" id="jhpbio">
+
Xuexuan Zheng, Xinzhe Li<br>
-
<h1></h1>
+
Introduce the lambda phage’s binding receptor on the membrane of the E.coli. The site is too complex that we can hardly make use of it.<br><br>
 +
Sitan Zhang<br>
 +
Introduce the system of Quorum Sensing<br><br>
 +
Yangming Yue<br>
 +
Introduce a novel method for suicide: lysozyme + holing + anti-holin<br><br>
-
<div class="clear"></div>
+
Jinxin Wang<br>
 +
Introduce a type of plasmid keeping system. In this system, three necessary genes in the genome of the bacterium are knocked out and are installed into the plasmid. The bacterium can survive only if it keeps the plasmid. <br><br>
 +
 
 +
</p>
</div>
</div>
-
<div class="bio" id="yywbio">
 
-
<h1></h1>
 
-
<div class="clear"></div>
+
<div class="bio" id="xzlbio">
-
</div>
+
<h1>13th, May</h1>
 +
<p>Place: Room 363, Life Science building
 +
</p>
 +
<p>Instructor: Mr.Hong
 +
</p>
 +
<p>Recordist: Xinzhe Li
 +
</p>
 +
<hr />
 +
<p>
 +
I.Antisense-RNA<br><br>
-
<div class="bio" id="xlzbio">
+
Li Xinzhe:"kissing complex"<br>
-
<h1></h1>
+
Instruction of the loop-loop interaction between RNA which called "kissing complex".  This structure may improve the effciency of the combination of RNAs.  However, this structure is a little difficult to design.<br><br>
-
<div class="clear"></div>
+
Chen Wuyang: Zcal<br>
-
</div>
+
The article presents five kinds of antisense RNA which are able to bind the mRNA of lacZ.  Only the first and the thired kind can repress the expression of lacZ.  This article assumes two essential tips about the design of antisense RNA: <br>
-
+
1)The binding site must include the RBS region. <br>
-
<div class="bio" id="yzbio">
+
2) The antisense sequence should not be very long.<br><br>
-
<h1></h1>
+
-
<div class="clear"></div>
+
Zheng Xuexuan: Paired Termini<br>
-
</div>
+
The article present a conclusion that the "Paired Termini" structure which appears at the end of RNA is extrem essential for the antisense RNA.<br><br>
 +
   
 +
Zhao Jieming: The repression of the phage gene expression by antisense RNA<br>
 +
There are four factors which determine the ability of repression of the antisense RNA:<br>
 +
1）The intensity of the transcrption of target gene<br>
 +
2）The amount of antisense RNA<br>
 +
3）The length of pairing sequence of antisense RNA and target mRNA<br>
 +
4）Whether the antisense RNA binds the RBS region of the target mRNA(There is also research which suggests the third factor isn't necessary)<br>
 +
The antisense RNA can target the coat protein gene, duplicase, maturation protein gene from the E.Coli. bacterialphage SP.  The maturation  protein gene is most suitable target region, for the reason that the production of this gene duplicates only once in the mature phage<br><br>
 +
Wang Song: The phage resistibility of lactobacillus mediated by antisense RNA    The author constructs antisense RNA of various length and find that the most effected one is the completely pairing and the longest one.<br><br><br>
 +
     
 +
II.Suicide gene<br><br>
-
<div class="bio" id="wyybio">
+
Yue Yangming: CcdB<br>
-
<h1></h1>
+
The main feature of ccdB protein:<br>
 +
1)repressing the DNA gyrase<br>
 +
2)stopping the synthesis of T7 polymerase of E.Coli, repressing the synthesis of DNA<br><br>
-
<div class="clear"></div>
+
Chen Wuyang: Lysis(the iGEM work of Singapore09)<br>
-
</div>
+
Feature: Partly destroy the plasma membrane of E.Coli<br>
 +
Effinicy: They adopted QS as the suicide trigger, because of the limitation of their project.  This makes their data can not show the intrinsic characteristics.  But we can find the ODE and the parameters they used in their wiki page and the paper they issued.<br><br><br>
 +
 
 +
III.Quorum sensing<br><br>
 +
Fu Ting<br>
 +
Evaluate and select the QS part which is used by USTC-2009 iGEM team<br><br><br>
 +
 
 +
IV.T7 phage parts<br><br>
 +
Li Xinzhe:<br>
 +
There are only two kinds of T7 promoter parts we can use, but both of them are reliable.<br>
 +
T7 RNA Polymerase parts are abundant.  One of them is widely used by othre team.  Berkeley2007 used to construcate a T7 RNAP library, the five mutants in the library could be tested respactly to select the suitable one.<br><br><br>
 +
 
 +
V.The schedul of the future<br><br>
 +
20th,May~10th,Jun<br>
 +
Preparation for the preliminary experiment<br>
 +
1.Rough modeling(especially for the ccdB and Quorum sensing), and determine all the parts we are going to use<br>
 +
2.Prepare the competent strains, plasmid and all the parts<br>
 +
3.The protocal including all the details<br><br>
 +
 
 +
11th,Jun~20th,Jul<br>
 +
1.Assignment and start the experiments<br>
 +
2.Obtain the quantitative data<br>
 +
3.Set down the construction plan accoding to the preliminary experiment<br>
 +
1）agenda<br>
 +
2）protocal<br>
 +
Preform the preliminary experiment<br><br>
 +
 
 +
21st,Jul~30th,Sep<br>
 +
Preform the construction experiment，Obtain the quantitative data<br><br>
 +
And other work:<br>
 +
Regional Jamboree attendance fee<br>
 +
Track selection<br>
 +
Project abstracts<br>
 +
Team rosters<br>
 +
Safety questions<br>
 +
Project and part documentation due, including documentation for all medal criteria<br>
 +
BioBrick Part DNA due to the Registry<br>
 +
Wiki Freeze<br><br>
 +
 
 +
During the summer vacation:<br>
 +
design Wiki<br>
 +
Presentation：make PPT<br>
 +
Free show：design poster<br>
 +
 
 +
 
 +
</p>
</div>
</div>
 +
<div class="bio" id="jwhbio">
 +
<h1>20th, May</h1>
 +
<p>Place: Room 363, Life Science building
 +
</p>
 +
<p>Instructor: Mr.Hong
 +
</p>
 +
<p>Recordist: Jiawei He
 +
</p>
 +
<hr />
 +
<p>
 +
During this meeting,we discuss more details about our two different projects.<br><br>
 +
1.Anti-phage E.coli<br><br>
 +
A. Construction of genetic circuits to test the function of our parts<br>
 +
a.Inhibition and activation to pRM<br>
 +
pRM is activated by the protein cI. By adding IPTG, plac is activated, which leads to the production of the protein cI. With the existence of cI, pRM-RFP starts to work. Therefore, we can test the function of pRM by comparing the intensity of RFP with and without IPTG.<br><br>
 +
b.Repression to the translation of cro by antisense RNA <br>
 +
⑴.Design an effective antisense RNA:<br>
 +
i). Length of asRNA. Use all the sequence of cro, or cut some fragments of different length?<br>
 +
ii).Formation of secondary structure.<br>
 +
iii).Excluding the cleavage sites.<br>
 +
⑵.Construct the fusion protein cro-gfp. <br>
 +
⑶.Construct the genetic circuit:<br>
 +
By adding IPTG, plac is activated, which leads to the transcription of Anti-cro. We can test whether Anti-cro is able to repress the translation of cro by comparing the intensity of cro-RFP.<br><br>
 +
c.Function of cII<br>
 +
The promoter PRM can transcript to the left with the assistance of protein cII. In that case, antisense RNA of cro is transcripted and protein cI is produced. However, it’s difficult to test the function of cII.<br><br>
 +
d.Suicide——lysis<br>
 +
The gene lysis can cause the suicide of E.coli. Likewise, IPTG activates plac, producing cI to activate pRM. Then lysis is expressed and E.coli will suicide.<br>
 +
In case of the leak expression of plac, we can improve the circuit with the aid of promoter T7, which is activated only when T7 polymerase expresses.<br><br>
 +
e. AHL Quorum Sensing<br>
 +
To build an “alarm system” in E.coli, we also use AHL Quotum Sensing system. When some of the E.colis are infected by λ phage, they use AHL to “warn” the others.<br><br><br>
 +
 +
B. Parts to use:<br><br>
 +
BBa_I12006：activated by lamda repressor (cI) and repressed by 434 repressor (cI)；<br>
 +
BBa_I12007：activated but not repressed by the lambda repressor (cI)<br>
 +
toggle switch：（2011 USTC-China）<br><br><br><br>
 +
 +
 +
2.Code-circuit <br>
 +
We use three kinds of informational molecules: AHL, Ara and IPTG as “keys” to the code-circuit.Only when the three materials are added in the right order, GFP can express. Otherwise, the promoters below are activated and lysis expresses to cause the suicide of E.coli.<br>
 +
However, we need to consider how fast the three informational molecules can combine with the corresponding promoters seperately. Mathematical model is what we need.<br>
 +
 +
</p>
 +
</div>
 +
 +
</div>
 +
</div>
Line 377: Line 632:
<p class="level1"><a href="https://2012.igem.org/Team:USTC-China/methods">Methods</a></p>
<p class="level1"><a href="https://2012.igem.org/Team:USTC-China/methods">Methods</a></p>
-
<p class="level0"><a href="https://2012.igem.org/Team:USTC-China/achievements">ACHIEVEMENTS</a></p>
+
<p class="level0"><a href="https://2012.igem.org/Team:USTC-China/results">ACHIEVEMENTS</a></p>
<p class="level1"><a href="https://2012.igem.org/Team:USTC-China/results">Results</a></p>
<p class="level1"><a href="https://2012.igem.org/Team:USTC-China/results">Results</a></p>

Latest revision as of 14:38, 25 September 2012

GROUP MEETINGS

Date

  • 18th,Feb
  • 25th,Feb
  • 3rd, Mar
  • 10th,Mar
  • 17th,Mar
  • 24th,Mar
  • 7th, Apr
  • 14th,Apr
  • 6th, May
  • 13th,May
  • 21st,May

18th, Feb

Place: Room 363, Life Science building

Instructor: Mr.Hong

Recordist: Wuyang Chen

I. Discuss about our project:
1. SNS:
To complete this project is hardly possible. While it is likely to realize part of it, such as a system which is able to create and sense signals. But we need to confer our project some new meanings.
1) About making a signal disappear, according to Mr. Hong, we can add a rapid degradation label onto the signal.
2) We are afraid that the colony is too small to create an appropriate concentration gradient. According to Mr. Hong, it won’t be a big problem.

2. Solve the graph colouring problem using E.coli
1) In fact, we will only use E.coli to emit light in different colors, and all the algorithm for solving graph coloring problem is processed artificially.
2) We need to consult professors in Chemistry Department about constructing structures using ssDNA.
3) The scale of DNA strand is far too small than that of E.coli.
4) The fusion protein of Zn finger and the Ag43 would be very useful. We can think about how to make use of it.

3. Fatigue of specific stimulus
1) comK is a protein produced by bacillus subtilis and “competence” is a state of it. The possibility of turning into competence state is proportional to the amount of comK. We can substitute the protein involved in competence state for the protein we need.
2) The system can be used as a special lock.
3) How to make circuit one recover its function as quick as possible?
a) by means of protease?
b) by means of aptamer?

II. Set up groups to continue brainstorm

III. Make plans for the whole term.

25th, Feb

Place: Room 363, Life Science building

Instructor: Mr.Hong, Yinghong Lan

Recordist: Xingwen Chen

Continue brainstorming.
I.Three ideas we have put forward before:
1.Bacterio-SNS (Social Networking Services )
problem1.The signal molecules may exist for too long.
solution：Add a degradation tag to the signal protein for the cell to degrade it quickly.
problem2.How to construct a memory system?
There are several ways but which is the most suitable? problem3.Does CDK exist in bacteria?
We can't get it until we find a paper about that.
problem4.We should consider what kinds of result we will achieve.

2.Solve graph coloring problem with DNA and visualize the result with E.coli.
problem1.How to construct the complex DNA structure?
We can ask a professor who are studying this.
problem2.Bacteria are much bigger than DNA,thus the visualization may be impossible.

3.E.coli mimic specific stimulation fatigue
problem1.The idea will actually be quite boring if our purpose is only to mimic.
Think of something new which can apply the system.
problem2.The signal molecules may exist for too long and signals may influence each other.
Change the signal and consider some physical signals such as temperature and light.


II.New Ideas
Zhou Shan says:
1.Bacteriaphage therapy

2.Synthetic biology moving into the clinic

3.improve bacteria tolerance：
a)efflux pump
b)heat shock proteins
c)membrane modification
d)general stress respond
These are mostly achieved by scientists.It is wrong to repeat others unless we can come up with something new.

4.Some thoughts about nucleic acid aptamer
a) Aptamer itself can be substrate.
b) Two aptamers cna be combined on a single bead.


III.Professor Liu suggest we could do something on the basis of our work of iGEM 2011.
Luo Siwei, a team member of iGEM2011,says that if we must do as this,the only thing we can do is to make it more specific,which seems a little boring.And he indicates that the work last year is too complicated and lacking in aesthetics.


IV.About aptamer
We have the technology and we can start the SELEX now.But we have to work out a good application of it.


V.Tasks of the next two weeks
Direction 1.Come up with a new idea on the basis of the work last year.
a)What can we improve?
b)Think about uncompleted work.
c)Try to apply the signal and response system we came up with in the SNS subject.
d)Try to apply the circuits in the specific stimulation fatigue subject.
e)Read more papers,view previous works and get some inspirations.

Direction 2.Bacterio-SNS:
a) Learn more about toggle switch.
b) Find out if there is something similar to CDK complex in E.coli or B.subtilis.
c) Think about what effect shall we get finally.
Direction 3:E.coli mimic specific stimulation fatigue:
Think of an application of it.

3rd,Mar

Place: Room 363, Life Science building

Instructor: Mr.Hong

Recordist: Xi Chen

I.journal club
Chen Wuyang, who talked firstly, introduced a kind of marine photosynthetic bacterium Rhodulum sulfidophilum which can produces extracellular nucleic acids. These were found to be produced concomitantly with cell growth. However, the mechanism of this phenomenon had not been figured out, instead, someone hypothesised that it is the process of lysis of a subfraction of cells that makes this happen. After that, the speaker addressed that using engineered plasmids and the bacterium can make the production of extracellular nucleic acid under control which had been proved by anther paper.

After that Zheng Xuexuan gave us a presentation about nanorobot. This autonomous DNA nanorobot was controlled by an aptamer-encoded logic gate, enabling it show different function in different cues. What’s more this kind of device can be loaded with a variety of materials in a highly organized fashion.


II.Introduction of SELEX
We invited Chen Liang, a graduate student, to refer us some knowledge about SELEX( Systematic Evolution of Ligands by Exponential Enrichment).In sum, he talked about the definition about this technology and the main round of this method. Additionally, some examples also were taken so that we can comprehend in a deeper degree.

III.Discussion about the probability of the SNS the program someone raised in the previous meeting and proposed more questions remaining fixed.

10th,Mar

Place: Room 363, Life Science building

Instructor: Mr.Hong, Yinghong Lan

Recordist: Zhaoxiong Chen

I.Xuexuan Zheng：several ideas
1.Use lactobacillus instead of E coli as our new model for genetically engineering in order to avoid the potentially harmful outcome to human so that these technology could be used in the field ofmedicine safely.
Difficulty:
1)source of proper strain
2)synthesis of standardized part
3)money required
2.Membrane RNA
Difficulty:RNA in membrane is unstable, easy to desquamate and be degradated
3.Two directions of sifting aptamers:1.theosine homolog 2.other aptamers whose properties is similar to theosine

II.Sitao Zhang:PNA（Peptide nucleic acid）
Properties:
1.stable at high temperature
2.the binding between PNA/DNA strands is stronger than between DNA/DNA strands 3.Better recognition capability
4.stable in the presence of protease and DNase
Functions:
1.bind to DNA and prevent DNA from methylation
2.recognize DNA with specificity
Usage:genetic switch and sifting
Difficulty:
1.unbearable cost
2.can't be standardized

III.Xuyuan Gao:
1.Use the gene of Tase and attenuator regulated by tryptophan to establish a recording net.
2.papers about aptamers:a kind of aptamer,KMF2-1a,could bind to some special proteins in cell membrane,then it could be transported into cell by endocytosis

IV.Qian Li:
1.Optical Control:
1)Synthesis a special peptide,one terminal of which act as a ligand and is able to bind to cell membrane,the other terminal is a systeine whose residue,a sulfhydryl,could bind to hydrogel with the irradiation of laser.That peptide could act as a bridge to bind cells to hydrogel.
2)Central section of the peptide should be designed to be a special structure which could be cut off by UVL so that the cells binded to hydrugel could desquamate.
2.Ferritin：Ferritin is a kind of protein looks like a box,it could transport ferrum inside it in vivo.These boxes only unlock when ph is 2.0(5.0 in procaryotic cell),shut when ph reach 7.5.
Application：put chemical compounds in ferritin,let E coli carry them to the area where cancer cells live and growth,then unlock ferritin,release our medicines to kill cancer cells.

V.Wuyang Chen:
a paper about how HIV works in cell:Protein Nup358 plays a important role in the process when the viral RNA is transported into nucleolus to integrate with cell DNA
Idea:construct several aptamers that could bind to Nup358 in order to prevent cell DNA from being integrate with cell DNA.
Difficulty:Nup358 is also very important in normal physiological activity,our strategy may do harm to uncontaminated cells.
Some sulotion:construct aptamers that could bind to the special domain of viral capsid which is used for binding to Nup358

Four directions of our team determined：
1.Qian Li:New cure for cancer
2.Xingwen Chen:New cure for AIDS
3.Xuexuan Zheng:Neutralize the effect of alcoholic drinks
4.Xuyuan Gao:Recording net inside cell

17th,Mar

Place: Room 363, Life Science building

Instructor: Mr.Hong, Yinghong Lan

Recordist: Ting Fu

Progress on our four ideas:
I.HIV
Wuyang Chen:
1.Introduce the structure of HIV and their functions.
2.Introduce the process how the HIV absorb and invade the T4 cell.
3.Useful details from two papers:
"Capsid is a dominant determinant of retrovirus infectivity in nondividing cells"
"HIV-1 Capsid-Cyclophilin Interactions Determine Nuclear Import Pathway-Integrati"

Discussion:
1.Application of aptamer.
2.Prevention of the cellular membrane conjugation.
3.T cell conduct suicide when it feel infected.

It is not practical for the team to deal with HIV, however, we can simplify our idea which concerns the resistance against the virus, that is, the phage. One thought is that we make the promoter of our parts the same as the promoter of the virus expression system. When the virus invades, it switch on two set of systems: one binds the phage inhibiting its reproduction, the other makes the host suicide. This project can be applied to fermentation production.


II.Cisplatin
Qian Li:
Useful info from papers: a novel aptamer KMF2-1a is internalized efficiently and specifically to the endosome of target breast cancer cells.


III.Relieving alcoholism and nicotine
Xuexuan Zhen:
About the alcoholism: design a positive feedback, making the Bacillus acidi-lactici heavily express components to decompose the alcohol.
Key: a alcohol sensor, a positive feedback, application of QS.
About the nicotine: expressions of unpleasant smell or something absorbs and relieves the nicotine when nicotine is taken by people. Some bacterium lives on nicotine as carbon source or nitrogen source. The relevant genes can be used to relieve the nicotine directly.


IV.The gene toutine
Xuyuan Gao: a routine responses only when a substance is added twice.
a encoded routine: a routine reponses when the amino acid is added at the right order.


More papers: Jinxin Wang: a therapy on cancer, antimetabolitas which inhibits the dividing phase. Cai Li: an aptamer combines with hemoglobin.

24th,Mar

Place: Room 363, Life Science building

Instructor: Mr.Hong, Yinghong Lan

Recordist: Wuyang Chen

About the project of virus

I. Introduction of lambda phage
1. Construction and maintenance of lysogenic life cycle
Repressor CI is the only protein produced during lysogenic life cycle. Its functions are:
1) combine to OL: represses the expression of the gene downstream the promoter pL.
2) combine to OR1: represses the expression of the gene downstream the promoter pR.
3) combine to OR2: activates the expression from promoter pRM and maintains its own expression.

Transform into lytic life cycle
Cro is responsible for preventing the synthesis of the repressor CI and this action shuts off the possibility of establishing lysogeny. It has two effects:
1. It prevents the synthesis of repressor via the maintenance circuit; that is, it prevents transcription via pRM .
2. It also inhibits the expression of early genes from both pL and pR Cro achieves its function by binding to the same operators as (el) repressor protein.

II. Idea about our project
1. Promoter: same as the promoter in lambda phage.
2. Aaptamer: try to bind the receptor the phage uses to anchor onto the membrane of bacteria.
3. Genes for defence:
1) gene for suicide.
2) gene cI.
3) gene whose products can inhibit the expression of Cro.

7th,Apr

Place: Room 363, Life Science building

Instructor: Mr.Hong

Recordist: Xuyuan Gao

The main component of the “Gene passwords” in this conversation:

1.Introduce the rudiments of the attenuator:
1)The gene structure of the attenuator;
2)The essence of why attenuator can reduce the production of amino acid.(Take the tryptophan attenuator for example and advance a personal opinion)
3)The mechanism of how the attenuator can reduce the production of amino acid.

2.Improve the “Gene passwords” :
Use some step of one circular biochemistry reaction as the nodes for control.

14th,Apr

Place: Room 363, Life Science building

Instructor: Mr.Hong, Yinghong Lan

Recordist: Xuyuan Gao

The main component of the “Gene passwords” in this conversation:

The data of the ”Gene passwords” (Attach the lecture for the “Gene passwords”)Except me, I need two other teammates for this group. For this is an original project, and there is no former production that we can borrow, and we don’t have the parts that have been made out. Though the second part of the project is really difficult, we may complete the first second part of the project, but I’m not sure that we can complete it certainly. If you are interested in the project, you can join me.

6th, May

Place: Room 363, Life Science building

Instructor: Mr.Hong

Recordist: Wuyang Chen

Project of phage

1. Part for initiating transcription: pRM, BBa_I714889
2. Part for inhibit the lambda from turning into lytic life cycle: antisense RNA
This is the core of our project, we need to deeply survey this part.

Xuexuan Zheng, Xinzhe Li
Introduce the lambda phage’s binding receptor on the membrane of the E.coli. The site is too complex that we can hardly make use of it.

Sitan Zhang
Introduce the system of Quorum Sensing

Yangming Yue
Introduce a novel method for suicide: lysozyme + holing + anti-holin

Jinxin Wang
Introduce a type of plasmid keeping system. In this system, three necessary genes in the genome of the bacterium are knocked out and are installed into the plasmid. The bacterium can survive only if it keeps the plasmid.

13th, May

Place: Room 363, Life Science building

Instructor: Mr.Hong

Recordist: Xinzhe Li

I.Antisense-RNA

Li Xinzhe:"kissing complex"
Instruction of the loop-loop interaction between RNA which called "kissing complex". This structure may improve the effciency of the combination of RNAs. However, this structure is a little difficult to design.

Chen Wuyang: Zcal
The article presents five kinds of antisense RNA which are able to bind the mRNA of lacZ. Only the first and the thired kind can repress the expression of lacZ. This article assumes two essential tips about the design of antisense RNA:
1)The binding site must include the RBS region.
2) The antisense sequence should not be very long.

Zheng Xuexuan: Paired Termini
The article present a conclusion that the "Paired Termini" structure which appears at the end of RNA is extrem essential for the antisense RNA.

Zhao Jieming: The repression of the phage gene expression by antisense RNA
There are four factors which determine the ability of repression of the antisense RNA:
1）The intensity of the transcrption of target gene
2）The amount of antisense RNA
3）The length of pairing sequence of antisense RNA and target mRNA
4）Whether the antisense RNA binds the RBS region of the target mRNA(There is also research which suggests the third factor isn't necessary)
The antisense RNA can target the coat protein gene, duplicase, maturation protein gene from the E.Coli. bacterialphage SP. The maturation protein gene is most suitable target region, for the reason that the production of this gene duplicates only once in the mature phage

Wang Song: The phage resistibility of lactobacillus mediated by antisense RNA The author constructs antisense RNA of various length and find that the most effected one is the completely pairing and the longest one.


II.Suicide gene

Yue Yangming: CcdB
The main feature of ccdB protein:
1)repressing the DNA gyrase
2)stopping the synthesis of T7 polymerase of E.Coli, repressing the synthesis of DNA

Chen Wuyang: Lysis(the iGEM work of Singapore09)
Feature: Partly destroy the plasma membrane of E.Coli
Effinicy: They adopted QS as the suicide trigger, because of the limitation of their project. This makes their data can not show the intrinsic characteristics. But we can find the ODE and the parameters they used in their wiki page and the paper they issued.


III.Quorum sensing

Fu Ting
Evaluate and select the QS part which is used by USTC-2009 iGEM team


IV.T7 phage parts

Li Xinzhe:
There are only two kinds of T7 promoter parts we can use, but both of them are reliable.
T7 RNA Polymerase parts are abundant. One of them is widely used by othre team. Berkeley2007 used to construcate a T7 RNAP library, the five mutants in the library could be tested respactly to select the suitable one.


V.The schedul of the future

20th,May~10th,Jun
Preparation for the preliminary experiment
1.Rough modeling(especially for the ccdB and Quorum sensing), and determine all the parts we are going to use
2.Prepare the competent strains, plasmid and all the parts
3.The protocal including all the details

11th,Jun~20th,Jul
1.Assignment and start the experiments
2.Obtain the quantitative data
3.Set down the construction plan accoding to the preliminary experiment
1）agenda
2）protocal
Preform the preliminary experiment

21st,Jul~30th,Sep
Preform the construction experiment，Obtain the quantitative data

And other work:
Regional Jamboree attendance fee
Track selection
Project abstracts
Team rosters
Safety questions
Project and part documentation due, including documentation for all medal criteria
BioBrick Part DNA due to the Registry
Wiki Freeze

During the summer vacation:
design Wiki
Presentation：make PPT
Free show：design poster

20th, May

Place: Room 363, Life Science building

Instructor: Mr.Hong

Recordist: Jiawei He

During this meeting,we discuss more details about our two different projects.

1.Anti-phage E.coli

A. Construction of genetic circuits to test the function of our parts
a.Inhibition and activation to pRM
pRM is activated by the protein cI. By adding IPTG, plac is activated, which leads to the production of the protein cI. With the existence of cI, pRM-RFP starts to work. Therefore, we can test the function of pRM by comparing the intensity of RFP with and without IPTG.

b.Repression to the translation of cro by antisense RNA
⑴.Design an effective antisense RNA:
i). Length of asRNA. Use all the sequence of cro, or cut some fragments of different length?
ii).Formation of secondary structure.
iii).Excluding the cleavage sites.
⑵.Construct the fusion protein cro-gfp.
⑶.Construct the genetic circuit:
By adding IPTG, plac is activated, which leads to the transcription of Anti-cro. We can test whether Anti-cro is able to repress the translation of cro by comparing the intensity of cro-RFP.

c.Function of cII
The promoter PRM can transcript to the left with the assistance of protein cII. In that case, antisense RNA of cro is transcripted and protein cI is produced. However, it’s difficult to test the function of cII.

d.Suicide——lysis
The gene lysis can cause the suicide of E.coli. Likewise, IPTG activates plac, producing cI to activate pRM. Then lysis is expressed and E.coli will suicide.
In case of the leak expression of plac, we can improve the circuit with the aid of promoter T7, which is activated only when T7 polymerase expresses.

e. AHL Quorum Sensing
To build an “alarm system” in E.coli, we also use AHL Quotum Sensing system. When some of the E.colis are infected by λ phage, they use AHL to “warn” the others.


B. Parts to use:

BBa_I12006：activated by lamda repressor (cI) and repressed by 434 repressor (cI)；
BBa_I12007：activated but not repressed by the lambda repressor (cI)
toggle switch：（2011 USTC-China）



2.Code-circuit
We use three kinds of informational molecules: AHL, Ara and IPTG as “keys” to the code-circuit.Only when the three materials are added in the right order, GFP can express. Otherwise, the promoters below are activated and lysis expresses to cause the suicide of E.coli.
However, we need to consider how fast the three informational molecules can combine with the corresponding promoters seperately. Mathematical model is what we need.

backtotop

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