Lablog

Week 1: 2012/11/20-2012/11/27

2012/11/21

IGEM 2013 team member selection has been successfully concluded! 18 students from the school of life science were selected from numerous candidates. Among them, 12 are juniors and 6 are sophomores. We believe our new team will achieve more than previous years!

Preparation for the coming 2013 igem competition get started after the close of 2012 igem competition. Several members of the 2012 team, including FanXiao, YangTianF, ZhuMeng, ShiXxiaoJ, LiJinY and SunXiaoC, had a meeting at 21th, November. During the meeting, they discussed about convening the first team meeting, exchanged views on the weak points of the previous project, and shared the lessons they learned. They concluded that:

1) the standard part must be used in the project--even if not used in the core part.

2) when coming to the importance of the project, the reasons shall be logically unflawed. Viable application should be included that benefits the society.

3) creativity \innovation is of great importance.

4) data and reference must be presented with the PART.

5) the MODEL part should be improved: members in the MODEL team should understand every detail in the MODEL including parameters, solvability and application. Ideally, MODEL should be a reliable guide to the wet experiments.

6) more efforts should be devoted in helping other teams.

2012/11/24

The first igem2013 team meeting:

1) introduction: Zhu Meng introduced basic information about igem competition and the time plan for the next 12 months.

2) task assignment:

a. presentation about the outstanding projects of last year:

There is 8 min for every presentation. The purpose is to know what igem is and learn from other teams.

b. brainstorm (not forced), email the ideas to other team members.

c. determine the proper time for next team meeting.

WEEK 2 2012-11-26 to 2012-12-2

2012/11/30

LJY emailed teammates a lot of review articles on synthetic biology. So many to read…but all seems helpful~

2012/12/1

Zhu Meng got an idea and named it TRANSGENIC DETECTOR：At present, the growing variety of transgenic crops have resulted two types of people holding opposite views. Some approve the development of transgenic crops, believing it plays a good role in people’s health care. Contrary to this opinion is the view that transgenic crops have unknown side effects which may cause disaster. For whatever the consumer’s attitude is, s/he would like to know whether the food s/he is eating is transgenic or not. People who appreciate transgenic technology hope they eat the genuine transgenic crops, while people who don’t fears uninformed taking in of transgenic foods. Detection of transgenic crops, however, can only be done by professional biology companies now, which costs a lot of time and money for most ordinary people.

Based on the background mentioned above, if household transgenic detector can be constructed by applying synthetic biology methods on yeast, the prospects of detecting transgenic food in an easy way will be bright.

To accomplish this goal, combination of yeast surface display technology, the utility of transgenic plant’s selectable marker protein and the autoinhibition function of WIS to achieve cellular signal transduction is needed.

The second igem2013 team meeting:

1. Zhu Meng was chosen to be the leader of our igem2013 team and she seemed confused on how and by whom this decision was made :). Zhu said this decision would put a lot pressure on her. She modestly said she was not the best and to win this competition needed everyone’s efforts.

2. Zhu’s project of transgenic detector was discussed. Several flaws were found: 1) it is unknown whether autoinhibition system will work because its working depends on the character of the fusion protein. 2) The topic of transgenic food is too much sensitive in nowadays society.

3. Presentation of last year’s outstanding project (assigned during the last team meeting).

Everyone has done a good work! And the Q&A part is very inspiring. All these showed that our teammates had understood what igem is and what we should do in the following months. We were impressed by how simple but functionally powerful the winning team’s project is, and how scientific and complete the second team’s project is.

4. Considering that every track in igem has its own feature, presentation tasks were assigned to 5 sophomores. Each of them will chose a representative team to show what each track deals with.

Yang Qian: Best Foundational Advance Project: Carnegie Mellon

Xie Hengyi: Best New Application Project: LMU-Munich

Hou Lingfeng: Best Manufacturing Project: Utah State

Li Tianyi： Best Software Tools Project: Johns Hopkins-Software

Lu Xinping：Best Information Processing Project: Tokyo Tech

WEEK 3 2012/12/3-2012/12/9

2012/12/3

Junior members have a small meeting. They discussed the arrangement of next team meeting. Fan Xiao mentioned that he got an idea about typography. And they all agreed this was the right time to discuss project design with instructors.

Discuss with Pro. Chen: Fan Xiao, Wang Yuning, Huang Yanlan.

Discuss with Dr. Dai: Shi Xiaojing, Fan Xiao, and Zhu Meng.

2012/12/7

The third igem team meeting:

1. 5 sophomores gave presentations on the projects of best track. (5 min for each project, including an introduction to the project and comments)

Yang Qian: Best Foundational Advance Project: Carnegie Mellon

Xie Hengyi: Best New Application Project: LMU-Munich

Hou Lingfeng: Best Manufacturing Project: Utah State

Li Tianyi： Best Software Tools Project: Johns Hopkins-Software

Lu Xinping：Best Information Processing Project: Tokyo Tech

2. Yang He and Shi Binbin :) explained how to construct model. But most of us feel it is too professional to understand. Calculus…Multivariable calculus…oh, I got a headache…

3. Zhu Meng systematically introduced the features of yeast, classical signal pathways in synthetic biology, basic technology and application in igem competition. We find it may be not as difficult as we thought to manipulate yeast cells.

We are inspired by all the ideas. It will be definitely exciting to use yeast as our function system~~

WEEK 4 2012/12/10-2012/12/16

2012/12/12

Dr. Dai shared with us his several interesting ideas on the project:

1. As yeast is a eukaryotic system, it might be modified to have partial functions of lymphocytes. One possibility is that it might synthesize phages which could kill bacteria or synthesize antibodies to collaborate with pre-existing immune system, or it might be modified to recognize and phagocytose bacteria as a macrophage. Cool idea as it is, however, in my known case, no paper directly related is found. The overall mission seems to be tough, but making small achievements are promising. At least we may focus on recognition of pathogen by yeast.

2. Cell-communication between tumor cells in drosophila might be investigated and transplanted by yeast system. This is a grand new aspect that few papers are published but valuable materials are available.

2012/12/15

Li Xiaoyi got an idea: using RNA to simulate GFP

We all know that it is GFP protein’s fluorophore that give GFP the ability to produce fluorescence. The paper I sent to everyone is about screening an RNA named Spinach. This RNA has a special secondary structure. Its blue part, named aptamer, can bind to different substrate as we modified its DNA sequence. When add chromosphere, fluorescence can be observed.

Pros:

1. The method commonly used can only use fluorescent protein to reflect the change of protein level in cell. This new method can reflect the level of substrate.

2. The substrates of aptamer are usually ADP or SAM. We can design different substrates but needs searching other reference.

3. Easy to design. We can alter the stem-loop structure of the RNA in ways we want.

4. Classical synthetic biology methods can be used to control transcription.

5. The intensity and stability of the fluorescence are good.

6. There are lots of HBI analog. Adding different chromosphere can produce different color.

Cons:

1. If merely used as an output signal to detect expression, GFP system would be better.

2. Specific project is still unclear. I don’t know what the upstream regulatory mechanisms is. Also the whether signal transduction depends on protein or small molecular is unknown. Don’t know if it will help.

3. If want to use it in painting pictures or changing color, chemical molecular must be added during the process. How to achieve automation is a big problem.

4. This system can work in E. coli, but in vertebrate is unknown. Don’t know whether it will work in yeast.

WEEK 4 2012/12/17-2012/12/23

A large wave of exams attacking…followed by the happy Spring Festival! So for the next three weeks, the team meeting are canceled. We shall still focus on brainstorm and everyone will give a presentation on their brainstorm ideas.

WEEK 5 2012/12/24 -2012/12/30

NR

WEEK 6 2012/12/31-2013/1/6

NR

WEEK 7 2012/1/7-2012/1/13

2013/1/9

Zhu Meng, Fan Xiao and Yang Tianfang talked with Pro. Sun about our project. Pro. Sun suggested the visualization should be strengthened.

2013/1/11

The fourth igem team meeting:

Lots of good ideas! But further reference is needed to confirm the details. Several ideas were picked up for further development:

1. Timed-release drugs from Huang Yanlan

Time controlled. Capsule Storage microorganism which can release drugs opportunely and can sense the concentration of the drug.

Blood sugar may influence our metabolism. Release insulin to regulate it.

Help with jet lag.

2. Immunological recognition (mycoplasma) by yeast from Li Chaochang.

Direct way: Gram positive bacteria can release exotoxin; add exotoxin receptor on yeast (react when dimerization)

Indirect way: promoter array

3. Cells enriched protein from Wang Yuning

Vertebrate protein, non-secretive protein in prokaryotic system can be difficult to collect. Enrich in membrane system.

Lipid-dependent (PI4P), PI4P binding domain can enrich in TGN.

Secretory vesicle enrichment. There is some inhibitor which can allow vesicle synthesis but not secretion.

a: synthesis α: Secretion

Synthetic ester raft.

4. Complete remove of aflatoxin from Zhu Meng

Washing by water cannot get rid of all the aflatoxin; fungi, yeast cell wall can adhere to aflatoxin molecular; antigen-antibody reaction.

5. Yeast combined signal system from Sun Xiaochen &Yang Tianfang &Fan Xiao

Yeast can recognize lots of signals. Signal integration and processing can be achieved by mating, and output other kinds of signals.

6. Reconstruction of beer yeast from Li Tianyi.

1) Reconstruction yeast

Sober up (suitable temperature, pH and environment), Vitamin B6

Ethanol - acetaldehyde - acetic acid, the second step is always different in different people.

2) a, α

Function when diploidization.

Now we should focus on literature research.

WEEK7 2013-1-14- 2013-1-20

2013/1/14

The feedback got from instructor was that yeast combined signal system project and complete remove of aflatoxin project are interesting and can be further developed. But yeast system is challenging and no one can insure it will work.

2013/1/15

Sun Xiaochen and Zhu Meng talked with Dr. Dai about the two projects. Dr. Dai felt the designs are good. Then he shared two ideas with us.

Winter Vacation (2013/1/21~~2013/3/5)

2013/1/25

We received invitation for across the strait igem communication from NCTU. Sophomores are willing to attend the meeting, but ticket reimbursement by the school is a problem.

2013/2/2

What a surprise~ Yeast operation is quite a tough issue. Most of the members who do the lab work frequently amazed by some additional test steps used in yeast clone selection. But the toughest thing is, the materials in kit cannot work properly...the clone of some fragments in yeast should be done earier.

2013/2/3
the pre-experiment is still going on. delighted by the news that members have almost master the skills. the method to test the efficiency of yeast expression system is to construct the promoter-GFP system. First of all, the target of testing is the promoter TRR, which should be used in AFT cleaner idea as well as the convertiable detector. so called " two birds killed by on stone". Overlap PCR should be used. the primer is designed by some members.

2013/2/4
some members just come back from australia, welcome to Beijing~ Beijing huanying ni~ however, the lab is nearly closed because of the spring festival holiday and everybody have to go back home to gathered with their families. So the pre-experiment will be stopped for several days. IGEM, see you the next year~(see to be such a long time = =)

2013/2/13

Yang Tianfang got an idea. He said that using yeast as formaldehyde purification device would be nice. Using promoter which can sense the presence of formaldehyde. Details should be further determined.

2013/2/16

Sun Xiaochen gave some comments on Yang’s project:

Good application!

To extend his idea, maybe we can design an air quality testing machine. Detecting things such as formaldehyde, CO, harmful gases caused by smoking, or even detecting PM2.5 by light absorption (this is hard to accomplish, too difficult to establish a sensor sensitive enough to do the job, just a thought…).

2013/2/23

The cross strait igem communication is unable to carry on for the school cannot afford our costs. And no invitation is received. Cooperation and communication would be thwarted by distance in igem…

2013/2/24

First team meeting~ everyone looks good. But we should take all the works seriously. So many works remain unfinished, so many…

2013/3/3

Tianfang introduced a team collaboration tool called Colorwork. One of the developer come and give us a brief introduction of how to use it. Seems useful~~

2013/3/4

JHU sent us the PART of carotene degradation enzyme. We can start now! Integrate it into the system first! But we heard that JUH will not send igem teams this year. Alas. Although every team wants to achieve something in the competition, the brutal reality of get funded is an essential problem…

2013/3/10

Registration is successfully done! Thus, 2013 TSINGHUA IGEM team officially found! Hope we can surpass the last team as we claimed in the introduction. Break all the limits!

2013/3/16

What a day, amazing TianFang was inspired by a guy called ZHU Junhao and was then wrote the words like this:( you guys can feel it~)

After discussion with ZHU Junhao and ZHAO Yanyan, novel ideas had occured to me, and an improved understanding of what we are supposed to do struck my mind. I felt nervous and passionate, as well as a little afraid. Well, as least we still have time to adjust the team and project. Go on bros and sisters!

What on earth happens here??? What is the idea? Wait, a feel of ominous appears….

2013/3/18

Okay… I submit.. A guy called Zhu Junhao inspired all of us and we then scrutizie our project again and found it to be too boring. Then we planned to change the previous idea and get a new round of brainstorming . Though the process might be annoyance as we don’t have too much time on it, since most of us will not at school during the summer time. But I believe it will be worth it, only everyone love the project we can indeed do something and achieve something.

Here is the sentences wrote by Yang Tianfang after the “propaganda” by Zhu Junhao:

(you guys can feel it)

Today’s meeting is the longest one, but I feel stirring, like found a passion that has lost for long. Thanks Zhu Junhao “ruined” our team meeting. Thanks everyone in the meeting. I reckon this is the real brainstorm.

At the same day, planned and organized by Yang Tianfang, we booked the Xuetang every night and promised to get together every night and discuss the idea we can find. We want a indeed Brainstorming. Nothing else but a blackboard, a pen, and several brains. We think about everything we can imagine, think every idea that just come up into our mind and look into it very carefully, think about the parts we can use and think about the whole system. Though the direction is obscure and not defined, believe nothing is impossible for the Tsinghua Guys.

2013/3/22

Okay~! Amazing, the brainstorming generate totally 5 ideas with certain details. Discussed with Prof. Dai, we then think about the best one to do. Several members like Xiaoyi and CC are working with the papers then can find and the others design the parts and systems.

At the same time, HP has several process ongoing, the first short movie is about to playing out. Several people discuss about how to yield the biggest effect on that and how to motivate everyone to watch. Anyway, the transcript of the movie is great, all is about the sex determination process. BTW, the movie is actually one part of the whole series, the goal of which is to propaganda the knowledge related to biology, especially the synthetic biology. Fabullous painting, creative transcripts and effects, believe they will be very popular!

2013/3/23

The registration fee has been successfully wire transferred to the IGEM HQ, Then we are allowed by law~(sounds like get married, but it was totally 18 people, oh my goodness….)

But on the other side, the time is quite limited, it has already been the end of March, but the experiments hasn’t been carried out, the idea is incomplete, the other parts like human practice still need further developed. And the most essential thing, the judging criteria must been changed this year. So bad… what can we do?

2013/3/25

Another important motivation meeting after the beginning of this new semester. Several changes within the team now com out. Firstly, team has been divided into several small groups, experiment, model, wiki, poster&design, transaction. The team leader is in order to be Xiaochen&Jinyang, Binbin, Tianfang, maomao, Xiao bro~ And we promised every meeting every group leader should report their progress as well as the plan for next week. I admit that all about the changes is to lazy myself = =..Also, the other changes come to the design of the experiment, worry about the achievement of the new project, we do the experiments for both of the two project, though it might waste a lot of resources, just in case we cannot done everything.

2013/3/31

Yuning Wang reported the process about human practice: the transcript for the first part of the series, the sex determination has been translated into English, and are ready to be “action”! Good~

Binbin has designed the model for the first project, looks very dedicate and complex, but scientific and considerated.

About the experiments, plasmid has been transformed into E.coli. but the strange thing is every time the colonies filled in the plate and cannot pick up the single colonies, The antibiotic chloromycetin might be the one to blame. Next time when meet the plasmid with anti-chloro, be careful!

The promoter which can sense the H2O2, the TRR promoter is transformed by XiaoJing, RNA isolation by Xiaoyi and Meng is going to be done next week, as the amazing Armillariella tabescens have arrived yesterday. But the protocol we have still seems to be strange and ridiculous. (Only use the potato to incubate the colonies? We are so worried about the contamination as well as the yield)

Brainstorming still need further doing.

2013/4/1
1. the annoyance appropriation request have been done today~WHOO~expect everything will be OK at least in this section~ or something will be in trouble
2. there is something wrong in the DNA sequence of anti-AFT in the published paper, fortunately, another fragments sequence displayed on NCBI is the alternative choice, though the published paper declare that their antibody have the better specificity~ Meng is checking the sequence and making the attempt to synthesis fragment

2013/4/2

The sequence for antibody has been submit to Dai Lab, as it do not have the template the synthesize in vitro…. All the restriction site has been mutated artificial. And standardized. Prey~~~

2013/4/3

The new process of the brainstorming. All the thing about is to make a portable test paper using fried Yeast. and take advantage of the mating process of Yeast haploid, to make a switch box which can sense different signals input and generate different output. Great idea~ Now the problem is how to sense the signals and which signals to output? First idea is to be the pathogenic microorganisms. But how to sense the pathogenic microorganisms?

Xiaochen has invstigated and consult such ideas with the medical school students. Replied to be several medicines can inhibit the growth of microorganisms by various ways, yet the testing is still the important and cannot solved issue. Okay, just change a little bit~ went to another thing might be the better choice. Or maybe not.

2013/4/5

New breakthrough! Though it was hard to detect the pathogenic by such as antigen – antibody reaction, or like directly interaction , or synthesize the new information pathway in yeast, there is actually a very simple way to do that! Detect the AHLs existed in pathogenic microorganisms! First of all, AHL has specificity and differed in different microorganisms. Second thing is, actually qurum sensing system parts are easy to get, so many teams in the previous years work with QS system, as well as Tsinghua Team~ Amazing, it sounds like the best candidate was just standing behind us….( Okay this is actually sounds horrible…)

2013/4/7

The bacteria used to clone the gene ADZT is still not here, somebody is going to be crazy about that. But anyway, waiting is the only way that can do. A new member is going to be join in this team, WANG Jun, from Lanzhou University, a senior student. Welcome!

2013/4/11

The JHU’s plasmids have been received! The genes for synthesize beta-carotene, I believe they will turn out to be worked as expected~Now the plasmids are passed to Tianyi and Tianfang for transformation to Yeast.

2013/4/12

Finally HP short movies was out with the first version!!

2013/4/14

Plasmids has been extracted by maomao, the beta-carotene plasmids sent by JHU~~

2013/4/15

A new human practice! Molecular cooking~

2013/4/15

The whole project design has been done by Jinyang~ totally divided into three parts, with test, RFP output, as well as final part deisgn. Now just wrap the finger and ready to do the experiments!

2013/4/17

New meeting:

AHL should be purchased. Assigned to Meng, Finally our team logo and several candidate design have come out! All of them looks very beautiful, a big yeast in the back~ The wiki will be done by a classmate of Xiaojing, he is actually the member of colorwork, Good~ professional Several AHL has been discussed about to use as example. AIP, luxR system, LasR system…. It seemto be LuxR still are the one to be chosen.

Now there are two things we need to discuseed about. First is the specificity, the second is how can the AHL be passed into yeast cell membrane?

The yeast powder has been accomplished by Tianyi. And it has been tested that the powder can be reserved under room temperature and natural environment for as long as two weeks. Good~

2013/4/18

The potato medium for incubation for amazing Armillariella tabescens has been prepared by Wang Yuning and Meng. But, it smells very strange…..

2013/4/22

New meeting. But every one has been extremely busy these days, several people attend….

Progress is , the Human practice section runs very well, we plan to put it on the website, but don’t know if it will works.

2013/4/23

RFP, LUXI, LUXR has been synthesized by PCR and works well.

2013/4/29

Tianfang has consult the medical center located within the medical school about the Microarray, which is aimed to test the promoter of yeast and the comparable change of expression downstream of each promoter. It sounds like the only problem is none knows how to analyze the data, and the other thing is ,how to prepare the sample? And , what about the expenses? It must be very expensive…. Okay, the slovinia way may not work well under our circumstances…

2013/5/5

First experiment for overlap PCR has been done, but failed to have the obvious bands and obvious expected location. Okay, try another time.

2013/5/15

Yesterday I constructed RFP-413 and RFP-423. And today I did the bacteria liquid PCR to choose positive clones. I chose three positive clones per construct to extract plasmid on May 16th. There are 3 things to do for further work. First, do the sequencing to make sure we get the right sequence. Second, express RFP in yeast to see if it works. Third, continue to construct promotor to the vector.

2013/5/16

The GPD promoter is very hard to get? Terrible.. maybe we can change to another promoter? But we really do not know about it in the previous time. Okay, but the truth is, nearly everyone will be gone at the end of the June. So what can we do? The process of the experiments, the deadline is coming!

2013/5/17

Another plan comes out, replace GPD with TEF , another strong promoter of yeast. I think it can work this time.

2013/5/21

The negotiation with Ji Sun, the administrator of colorwork goes well. He can do the wiki design during the July~

2013/5/23

A lot of experiments need to be done!!!

1. Fluorescent protein: five totally, amplify by PCR, cut by BamHI and EcoR, ligate to 414 and 424. (Charged by Tianyi); 2. Clone CYC terminator and GPD promoter, ligate to pRS413-mRFP and pRS423-mRFP (charged by Xiaoyi); 3. Clone LasR and CYC-overlap, ligate by overlap PCR, cut by SpeI and BamHI, ligate to pRS413/423-GPD-mRFP-CYC. Clone LuxR and CYC-overlap, ligate by overlap PCR, cut by SpeI and BamHI, ligate to pRS413/423-GPD-mRFP-CYC. The cardinal work of this week is overlap PCR. 4. Cut rtTA by BamHI and EcoRI, ligate to pRS413/423-GPD-mRFP-CYC. Clone TetO, ligate to the fluorescent protein plasmid. (Charged by Jinyang) 5. PCR of Luxl has been done. Now need PCR CYC, cut by EcoRI, BamHI, BglII, XhoI, ligate to the mRFP plasmid (this could be done first. Xiaoyi and Chaochang can first finish this before the primer of GPD comes, then add GPD and CYC). Everyone give a hand!!!!!!!!

2013/5/24

Yeast Induction protocol

Colonies are picked from petri dish and suspended in suplemented SD media.
Incubate overnight
Resuspend in YPD8%
Incubate overnight to 5 OD.
Resuspend in YPRE
Incubate for 8 to 24 hours
Add a final concentration of 0.3 mM of H2O2
Measure OD and fluorescence intensity.

2013/5/26 From Tianfang

I have asked all the workers in the lab. None of them said there is vector in the lab containing promoter and terminator. They add promoter with target gene when construct the vector. So I decide to construct the vector by myself, including pRS413,414,415，pRS423,424,425, which may be useful in our project. Add promoter and terminator to the 6 vectors’ MCS region. I chose another strong promoter TET promoter for we don’t have GPD promoter in the lab. The terminator is still CYC. I have ordered the primer for TET promoter. May finish the construction of the 6 vectors containing promoter and terminator this week.

2013/5/27

Please have a look of our Human Practice Movie!~~

http://www.bilibili.tv/video/av573439/

2013/5/28 From Binbin

There is an article in Nature: Synthetic analog computation in living cells (http://t.cn/zHwZsLG). It applied the AHL-LuxR model. Some model parameters in the supplementary information can be useful. Its model is instructive.

2013/5/29

We take a photo as a team today! So many happy faces~ happy smile~

2013/6/1 From Tianfang

Today we followed Valencia’s protocol, use 30mM and 100mM H2O2 to treat transformed yeast. But we didn’t see fluorescence enhanced compare to control. So I decided to exam the vector to see if any relevant promoter fragment is missed. And I will construct many other vectors containing TEF promoter and TEF terminator. I’ll send the plasmid map to you and start PCR tomorrow.

Sad story: the lad do not have CYC terminator…it’s all my fault. I sent Jinyang the wrong plasmid map. Now we have plan B: 1. Wait for the construction of vectors containing promoter and terminator. 2. Test without terminator. Some of the senior members in my lab do this to express protein and it works.

2013/6/2

The design of the team clothes out !!! Great appraises for Maomao!

2013/6/3

Tcyc has been synthesized by the template of E.coli~

2013/6/4

Now most of the people have sense the dangerous smell~~They have decided to work something out , some people have already going to do the experiment in the mid-night, Okay, don’t push yourself too hard….Jinyang, Xiaoyi, CC, WANGJun Meng, good luck~~

2013/6/5

The design of Wiki(accurately described as the style of wiki has been finally worked out by Hou Lingfeng~~ Looks nice~~)

Also, he draws some pictures of the team members, very lively~~

2013/6/7

The first member has left the team, went to America good luck…

2013/6/8 From Tianfang

Didn’t receive any reply from Valencia after two emails. We’re going to ligate the three aflatoxin synthesis genes to one pRS413 by Golden Gate Assembly. Let the yeast get red first! We will focus on this before the final exam. Hum~

2013-6-20 From xiaoyi

PRS413/423-GPD-mRFP ligation and following steps:

1. I picked up eight clones after transformation; inoculate into 1mL culture for 4h; PCR, but has not do gel electrophoresis

2. Next is clone identification, including sequencing the plasmid to see if GPD is successfully inserted.

3. Plasmids extraction, transformation to verify the function of GPD.

Plates and cell culture are put in the 4℃ refrigerator right to the lab door, the top reads igem, on the left.

The most worried time comes, all the people going to go…

2013/6/21

Finally, one people, Hengyi decided to cancel the team. Because he is very busy and cannot squeeze the time for IGEM… So bad..Okay, good luck, guy~

2013/6/25

Here is some thoughts from Meng about Model:

PART I: Stimulation of mRFP expression in Yeast system

MODEL：AHL enter the nucleus and accumulate-->

LUXR infusion protein expression--> AHL and LUXR+AD DOMAIN active downstream gene express (two parameters are needed here, one is LUXR combine PLUX，another is AD combine MINI CYC PROMOTER), mRFP express, turn light，use time as parameter draw the MODEL prediction line(start from adding AHL as t=0, beginning AHL concentration influence on reaction time t should be included)

The fluorescence of mRFP measured in experiment, (to test the goodness of fit to the model line)

QUESTIONS: Which of the entering and accumulation of AHL in nucleus and LUXR expression is the rate-limiting step? If LUXR is the one, then parameter determination of LUXR fusion protein expression using t as a variable detected by western is needed.

Control MODEL: (don’t know whether non AD DOMAIN LUXR recruiting POL II can be described by parameters?), if can, use this to replace the mRFP expression line fitted by parameters of AD DOMAIN.

PART II: considering the fluorescence proteins expression of TET system（to be continued....)

2013/7/5

Finally, all the junior members board on the flight to different countries. What about IGEM? But when think about the good aspect, all the sophomore works very hard, it still leaves some hope..

2013/7/12

Plasmid (2204bp) that containing LuxR synthesized by Zhu Meng has given to Lu Xinping, 35ng/uL. We’ll design, share and debug all the constructed plasmid maps as soon as we can and ligate to the pRS system. Another sad story is that we seem missed the igem team registry time and I’m not in our team T-T. I’ll send an email to igem headquarter to see how to solve.

2013/7/19

Tianfang constructed the LuxR expression part: GPD promoter + LuxR (containing VP16, NLS, and CYC mini promoter) + mCherry (highly expressed red fluorescence protein in cell) + CYC terminator. The primer has ordered and when it come we’ll start!

2013/7/30

Finally we chose the track of health and medicine.

2013/8/8 From Tianfang

After two week’s effort, we successfully inserted the TEF promoter + LuxR fragment mixture + mCherry fluorescence report gene into the vector. Results of enzyme digestion shows that all are correct. Sequencing result is still waiting. To accelerate, we’ll extract the plasmid and transform into yeast to test the fluorescence protein expression. Thanks Li Tianyi who done most of the work efficiently. We’ll start together tomorrow. It’s a shame that sophomore has junior beat~

2013-8-10

Sequencing results show that pTF4 constructed by Li Tianyi are correct. Next is transformation. At the same time add AHL to see if red fluorescence express, and optimize conditions. But first, Zhu Meng, where had you put the AHL!

2013/8/23

Finally the yeast has grown up on the plate, there are certain difference of the mCherry signal before and after AHL, good, but they still not very high. When look back to the paper we designed and based on, it seems the concentration of AHL should be increased. Okay, work again.

2013/8/27

The statistic data has work out, and the difference between control and AHL group has qualified. Difference generate from not only numbers of cells but also the signal intensity of the cells.

2013/9/7

【resume work team meeting and work plan】

1. Two emergent igem official deadline:

1.9.18 PARTS received by MIT (Tianfang, Xiaoyi, Xiaochen must finish before 9.9)

2.9.28 Project and part documentation due, including documentation for all medal criteria( the most most most important deadline，all our work must be done before this）

2. Work plan next week:

Hou Lingfeng ：1. Repeat the inducing yeast experiment using AHL, three different AHL concentration（depends，notes：Wang Jun asked 3 AHL of different LUX from Peking University, so we don’t need to consider the dosage of AHL.），temporal resolution should be as high as possible (N hours as interval)，insure the viability for drawings 2. Increase AHL permeability by PEG, PEG concentration 2-3. 3. Count the light strength of 50uM yeast, ask Fanxiao for help.

Yang Tianfang：pTF5, pTF6 construction and efficiency detection

Li Tianyi：Transformation of fluorescence protein to yeast, take photos, pTF powder, send the protocol to Zhu Meng.

Li Xiaoyi：WB detect LUXR expression, negative control, positive control. Yeast with flag fusion protein transformed can be provided by Yang Tianfang; WIKI experiment part.

Li Chaochang ：important CONTROL vector construction：luxR+plux；luxR+plux+miniCYC, primer part.

Wang Jun ：PF：SpeI restriction site, flag tag (if too long yhen can give up），PR：BamHI restriction site.

Sun Xiaochen：FCM, help Tianfang construct the vector, manuscript

Li Jinyang：Manuscript.

Fan Xiao : WIKI, team, acknowledgement part

Huang Yanlan : team close design，poster design (style, overall arrangement, upload to COLORWORK for discussion), design of souvenir, LOGO position (contact Zhao Yanyan for production).

Lu Xinping ：Visa, documents

Zhao Yanyan ：stuffs about going to Hong Kong，team close and souvenir (contact Huang Yanlan for design )

Shi Binbin ：Wiki and debug, model and wiki model part, present model part next team meeting

Shi Xiaojing ：Safety design.

Wang Yunning and Yang He：HP card about our project, in the same style as before, understandable, WIKI HP.

Yang Qian ：NOTEBOOK translation

Zhu Meng：TJU team GC-MS，experiment manuscript，notebook，other stuff

Finally，experiment team meet at the ground floor of medical building at 21:00 every day.

Counting down 20 days. Come on！ Try our best, leave no regret！

2013/9/27

We have completed almost all the work.

Wiki: nearly finished.

Pamphlet: done.

Poster: being done.

Experiment: several complementary experiments need to be done.