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Team:USTC-China/groupmeetings
From 2012.igem.org
GROUP MEETINGS
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.
