Team:XMU-China/brainstorm

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MINUTES OF THE FIRST BRAINSTORM MEETING

Place: Chemistry Building 104.
Time: 7:00 p.m., March 10th and 11th, 2012.
In attendance: Zhaoshou Wang, former and current members of XMU iGEM team.
Aim: To discuss the feasibility of all the ideas put forward by members.

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Group1: Regulating Cell Regeneration

Speakers: Muxin Yu, Ruosang Qiu, Yunxin Long

1. –Muxin Yu

Cell aging is related to telomerase. Telomerase may help supply a gap of the DNA synthesis mechanism. Besides, she described some research about cancer cell activation.

2. –Ruosang Qiu

Expands the project of 2011 XMU iGEM team.
a) Using AiiA to restrain AHL synthesis.
b) CcdA and ccdB: If ccdA>ccdB, the function of ccdB is restrained; If ccdA<ccdB, ccdB will inhibit DNA gyrase.

3. –Yunxin Long

The concept of rehabilitation, stem cell, IPS cell, cell reprogramming. And there would be many obstructionusing stem cells or reprogamming cells to perform iGEM experiments.

Conclusion:

     Considering our experiment condition, the second idea is the most practical. To control cells density, we can use ccdA to restraint ccdB and then release the DNA gyrase astricted by ccdB. Compared with site-directed mutagenesis and sequentialermr prone PCR, controlling cells density in that way would be more labour-saving.

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Group 2: Bioscreen

Speakers: Qingshu Wu, Yizhen Yan, Yuan He

1. –Qingshu Wu

Literature summarize.
a) Circuit with Boolean logic: simple circuit → intricate circuit.
b) About signal molecule.

2. –Yizhen Yan

a) Binary transfer → encoder → nand gate → digital tubes.
b) Density display device: A/D converter → encoder→ digital tubes. Special difficulties: Constructing an operational amplifier.

3. –Yuan He

a) How to decompose fluorescent proteins in order to recycle the displayer device? One of the ideas is to use pigment to stimulate fluorescence, and then add some enzymes to decompose it.
b) Cells immobilization → embedding cells.

Conclusion:

Some intricate circuit may be too long to express. In this case, connecting expressive gene with promoter in series or in parallel can help.
To construct recycling fluorescent device, fluorescent proteins should decompose themselves. How can we attain that?
After studying a lot of literatures, members knew more about biocomputer and how to transfer signals in biologic units. Yan drew a logic circuit simply which turns input signals (1 or 0) into corresponding numbers (1-9). In this way, our goal of constructing a display device will be easier to reach. To fix the fluorescent cells, they primarily decided to embed cells in special tubes.
But how to switch the number showing on the screen, namely how to decompose fluorescent proteins is the primary problem. Group 2 and Group 7 have started to find a solution to this problem.

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Group 3: Constructing Engineering Bacteria to Produce Cellulose

Speakers: Chaoqun Hu, Xinzhu Tong

1. It's hard to make use of cellulose.
2. Cellulose — endoglucanase, exoglucanase, β-1,4-glucosidase.
3. The project of University of Edinburgh in 2007: Intracellular proteins are not able to excrete by themselves. How to solve this problem?

To do research about:

a) Ways of cells secreting proteins.
b) How do original cells excrete cellulose?
c) In nature, how do living beings utilize cellulose?

What are the advantages of this plan?

Conclusion:
     After studying literatures, the members of Group 3 learned about ege gene and Bgat1 gene that published in 2012, which can be used to produce cellulose. Generally in the process of producing cellulose, some enzymes must be added for lysis, and then release cellulose out of cells.
     The members of Group 3 hoped to utilize the device controlling cell density which was constructed last year, and exchange ccdB to another gene which will let cells themselves decompose. Thus our engineering bacteria will not only produce cellulose with quorum sensing but also release it out of cells. Besides, the members of Group 3 are trying to construct a secreting route way and hoping to produce cellulose without cell disruption.

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Group 4: Photosynthetic Bacterium (PSB) Utilization

Speakers: Zhao Ma, Hong Sun, Youbin Mo
1. –Zhao Ma

a) Photosynthetic bacteria.
b) Mechanism of photosynthesis of purple bacterium.
c) The case of California Institute of Technology in 2011.

2. –Hong Sun

a) Cyclic photophosphorylation.
b) Nitrogen fixation.

3. –Youbin Mo

a) Metabolism of acryl amide.
b) Utilize of light energy: bacteria → amylase/protein → energy.
c) Finding usable bacteria.

Conclusion:
     Group 4 firstly introduced photosynthesis and its 3 major metabolic pathways. It's arduously to clone these pathways, so they are considering constructing some Biobricks, which help to transform the organics that photosynthetic bacteria cannot utilize to another matter that can be utilized. Thus some organics can be decomposed by our engineering bacteria.

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Group 5: Utilizing Engineering Bacteria of Quorum Sensing (QS) System to Increase Natamycin Production

Speakers: Rong Fan, Qian Liang, Xinyi Yao
1. –Rong Fan

a) Mechanism of bacteria quorum sensing (QS).
b) GBL (gamma-butyrolactone) signaling molecules.
c) About natamycin.

2. –Qian Liang

a) Regulatory genes — pimM and pimR.
b) High-efficiency production of natamycin.

Key points:

1) Is it viable for mass-producing?
2) Negative feedback and positive feedback.
3) Association of quorum sensing and pimM expression.

Conclusion:
     Group 5 mainly searched information about increasing the production of natamycin. But they didn't study about the intracellular expression of E coli. Besides, they didn't mention of the advantages of utilizing quorum sensing (QS) system to produce metabolite. Therefore, in the following days, the members of Group 5 should find answers to the two questions through studying literatures.

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Group 6: Secretive Expression of Recombinant Barnase by E.coli

Speakers: Que Huang, Zhibin Gu

1. Barnase, barstar, RNase.
2. Cancer cells → acidity → separation → death.
    Normal cells → basicity → non separation.
3. Cloning strategies of barnase gene.
    Are there any errors in high fidelity PCR and why?
4. Secretive expression and Kil-Km Secretive box

Conclusion:
     Barnase is a kind of RNase. It's toxic and will cause cells death. Barstar is the depressor of barnase. These two proteins interact and then neutralize the toxicity of barnase. The members of Group 6 introduced barnase and barstar from various aspects, such as their applied prospect. Whereas they didn't learn about any problem in the process of producing barnase, so we don't know whether synthetic biology can help to improve it. What's more, just to construct a Biobrick to produce barnase seems of not much value.
     After discussion, we suggested the members of group 6 to study about some difficulties in the productive process of barnase and try to communicate with Group 5, in order to find our advantages of utilizing quorum sensing. Besides, Huang introduced some information of Kil-Km secretive box, which can accelerate secreting proteins out of cells. So we hoped them to exchange information with Group 3 and help them to find secretive pathways.

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Group 7: Modulating the Concentration of Tricolor Fluorescent Proteins to Show Different Colors

Speakers: Zhenan Zheng, Sifan Wang, Jianzhao Chi
1. –Zhenan Zheng

Fluorescence resonance energy transfer (FRET).
a) Dual color FRET: Constructing fusion proteins is required.
b) Multicolor FRET: To detect base mispairing.

2. –Sifan Wang

Unstable fluorescent protein
Add peptide tail after GFP so that it can be identified and decomposed by intracellular proteinase. The key lies in controlling and modulating the fluorescence intensity and the degradation velocity.

3. –Jianzhao Chi

Function of some nano antibiotics: Modifying GFP's character.

Conclusion:
     Group 7 focused on the choke point of this project: The degradation of fluorescent proteins, i.e. how to switch from one color to another. This problem was mentioned by Group 2 as well. How to switch from number A to number B when constructing a bio-screen? After studying literatures, Group 7 put forward three probable proposals.

1) FRET mechanism.
2) Unstable fluorescent protein.
3) Utilizing nano antibiotics to regulate fluorescent protein.

     After discussion, we thought the second proposal is relatively viable. For the first proposal, mutual interference between yellow and blue lights is a hidden trouble. And the catabolic enzymes of nano antibiotics mentioned in the third proposal may be not easy to find.
     So the members of Group 7 are suggested to study further about unstable fluorescent protein and peptide tail. But all the three proposals are worth trying. And the study results of Group 7 can act as reference of the project of Group 2 — bio-screen.

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Group 8: Utilizing Aptamers Biobrick to Construct Biosensor

Speakers: Shuqin Hu, Mouzhe Xie, Zebo Hu
1. –Shuqin Hu

a) Aptamer: It is widespread used in electrochemistry field, but its application in biosome is still in initial stage.
b) Estrogen sensor.

2. –Mouzhe Xie

In vitro → in vivo
What's the relationship between the biosensor and synthetic biology?
a) Constructing probe molecules. Whereas it may be difficult to construct,     because single chains are not stable.
b) Application in microbe. Introducing unstable single chains may lead to     degradation.

3. –Zebo Hu

Feasibility analysis
a) SELEX ( systematic evolution of ligands by exponential enrichment )
b) Comparing aptamers with antibiotics     Better in stability and affinity; more extensive and more sensitive targets.

Conclusion:
     The members of Group 8 told us the definition of aptamer and biosensor, and introduced the project of Professor Zhaoyong Yang's group. Whereas they found that aptamer biosensors and synthetic biology had little conjunction. At present, aptamer biosensors are usually selected in vitro, such as attaching on surface of an electrode.
     If we want to do research about aptamer biosensors in vivo, there will be two ways:

1) Construct them in microbe, and let them transcribe single chains for making biosensor. But single chains are not stable. So this plan needs further research.
2) Select aptamers we need in vitro then transform it in vivo, meanwhile its function of sensor should be maintained.After discussion, we thought both of the two means are of great difficulty. The members of Group 8 can ask Teacher Wang and Professor Yang for help that seeking for the bonding point of aptamer biosensors and synthetic biology or Biobrick.