Team:Tsinghua/Notebook

Week1

Our team leader zhaoyu invited many experienced iGEMers such as zhangyun xiao ,liguanqiao and wangshiqi . They introduced to us the project the did last year ,and the basic concepts of synthetic biology and iGEM . Students in our school are very interested in this game and they ask so many questions so that the campus talk last almost for the whole afternoon .

Week 2 and Week 3

The team leader made a application form ,which focus on the applicants’ scientific research experience and some specific skills such as art design ,wiki design . We publicized iGEM through renren.COM which is the most popular website among Chinese students .Also at the help of director and monitor of all the class in life of school ,We sent our application message and email to every student . We left enough time for applicant to think about it .

Week 4

The team leader read all the application letter ,and reject some that don’t have time or treat the carelessly . then to let the applicant  get a deeper understanding of iGEM ,the leader hold two group meeting introducing more details about IGEM and synthetic biology . Six people of us also referred to one excellent team in previous years ,and introduced it to all . Through the official website of iGEM ,we learn a lot about iGEM quickly .

Week 5 and 6

After we know what iGEM and synthetic really is , we decided if we want to go on . Those left were divided into 6 groups . Then all people had brain-storm and chose at least 1 good idea . We have two weeks to choose a proper project and consult related papers to complete the project . We just require concept but not experiment design .

Week 7

We hold a group meeting for all group to present their idea . Each group give a 20 min presentation ,then questions were asked . we gave each other advise on their project , especially on feasibility . Most group thought out more than one idea ,so we had plentiful ideas .

Week 8

Our winter holiday last from week 8 to week 9 . During this period ,we researched different part respectively and communicated on the internet . In week 8 , we divided the project into different models and each was responsible for one . We researched in iGEM official website and many database to see if our idea has been realized .

Week9

The most important holiday, ie spring holiday was coming . We also need a rest after a busy term ,so we did nothing about our project this week .

Week 10

We wrote a report talking about our research result and share it with each other . Together , we debugged and modified our report . Finally , we finished a wonderful report including the background , significance, experiment design of the project .

Week 11

It’s the first week of new term , we all had kind of sundries to deal with . So we did nothing this week .

Week 12

We hold a meeting to decide our final project . We invited all instruct teachers and advisers to the meeting . First each team was given 20 minutes to present their idea and 10 minutes to reply questions . Then ,the teacher comment on every project . At last ,we voted for the most interesting project .

Week 13

We calculated the voting result and published the result .

Week 14

Considering the lab space and funds , we can’t all participate iGEM ,so the team leader talked to everyone to decided who can stay in the team . it cost one week to decide the team numbers.

Week 15 to week 18

During the four weeks , we designed the detailed experiment such as the part ,plasmid ,primer and so on . Then we referred to iGEM kit and different paper to see what we already have and what need to do . After that ,we consult the lab manager to see what we can do . According to the condition ,we altered our design again and again .

Week 19

In our design , we wanted form a well-proportionedbiofilm to communicate with each other and we also hoped signal reagent can spread properly . So we test different LB Medium concentration. We make gel of different concentration from 0.2 % to 2%. We found that 0.25% is proper .We use 0.25% Agar to make semi-solid LB medium in a dish. Then we plant the bacteria which has grown overnight onto the medium. We have a gradient of volume which is 100µl, 200 µl, 300 µl, 500 µl, 800 µl, 1ml, 1.2ml, and 1.5ml. Because the medium is semisolid, we can’t invert the dish. Consequently, water remains in the dish.

24hr later, we find that the medium has been covered with a layer of bacteria. 100 µl is enough and 800 µl is the most volume we can use.

Week 20

We clone gene lux I from 14-c in kit 1 of 2011 by pcr. then we digest lux I and plasmid pET-15b with NdeⅠand XholⅠ . Then we did gel extraction to get digested part ,and link them by T4 ligase .then we screen it by double digest .finally ,we got the target recombinant plasmid . We just use common protocol during the clone .

Week 21 and 22

To gota part that can express GFP at the induced of IPTG .We took 1-9H and 2-7I from kit 2011 ,transform them and extracted plasmid . then we digested 1-9H by EcoRⅠ and SpeⅠ ,2-7I by  XbaIand EcoRI.Since SpeI and XbaI has the same sticky end, we can link those two part ,although they were digested by different enzyme . We did this clone for many times ,we can get the recombinant plasmid ,but it just can’t work . We try again and again ,we just can’t observe the expression of GFP under the induce of IPTG . Then ,we sent the 1-9H and 2-7I to check sequence , only to found 2-7I is not as the description and the sequence was totally wrong . We had no idea but to change our design .

Week 23

Considering the error of 2-7I , we transform some part including 2-7E,lux I -15b ,RFP,FimE,we need and confirm them by pcr and order-checking . We found that 2-7E is wrong and the others are right .

   

Week 24

This week ,we clone gene FimE to pET-21a (XholⅠand BamHⅠ),RFP to 1-9H (XholⅠand NdeⅠ). the protocol is the common protocol .

We got the targeted plasmid .therecombinant plasmid of RFP and 1-9H can express GFP at the induce of AHL . We add AHL when OD600 reached 1.0 and we observed and recorded every one hour . We found that four hours after AHL was added red fluorescence can be seen by naked eye .