Team:Fudan Lux/Notebook

We summarized our recent brain storm result. At last we chose to make a light-signal oscillator in E.coli and possibilities to use nanotubes. The first thing for the next week is to find an efficient light sensor and a stable lighting part.

Discuss the establishment of the gene circuits.

Get lux without operon and promoter from k325909 by PCR. We used longPCR Enzyme and Overlapping PCR.

Measure the growth curve of dH5a transformed by lux with araBAD (k325909)

Transform tetR (C0040) from the kit. Transfer araBAD (I0500) and tetR (C0040) with RBS into pSB1C3 by 3A ligation. Transform it into dH5a.

Transfer tetR with promoter and RFP with ptetO as promoter (J61002) into pSB1C3 by 3A ligation. Transform it into dH5a.

Sorry to find the ligation is failed. But fortunately, we decide to change the reporter into GFP (I13522). Transform the plasmid from the kit into dH5a.

Design protein composed by lov domain and modified helix turn helix (abbr. lov-HTH).

Design protein composed by lov domain and tetR (abbr. lov-tetR).

Send the synthetic orders to the gene synthesis company.

Half of our members went to Tianmu Mount, Zhejiang Province. They spent a week there to participate a fieldwork organized by the Life Science Institute. During the fieldwork, they paid a lot of attention to a China’s specific firefly, Qiongyuying. They observed the behavior of Qiongyuying, especially the synchrony.

Our instructor and four of our members went to meet Richard J Roberts after the reward ceremony of COMBREX, a project to accelerate the functional annotation of prokaryotic genomes. They talked for about an hour and we presented our project to him. Richard advised that we should make our project more practical and functional.

After negotiating with the principal of volunteer teaching program in our institute, one of our members, with all members of Fudan_Lux’s hope and responsibility on his shoulders, went to Shangrao, Jiangxi Province to do the volunteer teaching.

Make an appointment with Li Jin, Hongyan Wang etc. to show our projects.

Receive lov-HTH in nonstandard vector.

Design primers to make clones of lov-HTH, and transfer lov-HTH with double terminators into pSB1C3.

Receive lov-tetR in nonstandard vector.

Do double digestion by E.coliI HF and SpeI, and transfer lov-tetR into pSB1C3.

Make an appointment with prof. Guoping Zhao, who establishes China’s first synthesis biology laboratory in Shanghai. He talked directly about the impropriation of our tumor project and the lack of application about our biowave project.

The double digestion and the ligation held us for more than 4 weeks!

In order to do the ligation more efficiently, we decide to do the ligation according to this table.

1.araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-GFP

2.araBAD-Rbs(0.3)-lov-HTH-Terminator-ptetO-GFP

3.araBAD-Rbs(0.07)-lov-HTH-Terminator-ptetO-GFP

4.araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO-GFP

5.araBAD-Rbs(0.3)-lov- tetR -Terminator-ptetO-GFP

6.araBAD-Rbs(0.07)-lov- tetR -Terminator-ptetO-GFP

7.araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-CFP

8.araBAD-Rbs(0.3)-lov-HTH-Terminator-ptetO-CFP

9.araBAD-Rbs(0.07)-lov-HTH-Terminator-ptetO-CFP

10.araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO-CFP

11.araBAD-Rbs(0.3)-lov- tetR -Terminator-ptetO-CFP

12.araBAD-Rbs(0.07)-lov- tetR -Terminator-ptetO-CFP

13.araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-lux

14.araBAD-Rbs(0.3)-lov-HTH-Terminator-ptetO- lux

15.araBAD-Rbs(0.07)-lov-HTH-Terminator-ptetO- lux

16.araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO- lux

17.araBAD-Rbs(0.3)-lov- tetR -Terminator-ptetO- lux 

18.araBAD-Rbs(0.07)-lov- tetR -Terminator-ptetO- lux

19.pLac-Rbs(1.0)-lov-HTH-Terminator-ptetO-GFP

20.pLac -Rbs(0.3)-lov-HTH-Terminator-ptetO-GFP

21.pLac -Rbs(0.07)-lov-HTH-Terminator-ptetO-GFP

22.pLac -Rbs(1.0)-lov-tetR-Terminator-ptetO-GFP

23.pLac -Rbs(0.3)-lov- tetR -Terminator-ptetO-GFP

24.pLac -Rbs(0.07)-lov- tetR -Terminator-ptetO-GFP

25.pLac-Rbs(1.0)-lov-HTH-Terminator-ptetO-CFP

26.pLac -Rbs(0.3)-lov-HTH-Terminator-ptetO-CFP

27.pLac -Rbs(0.07)-lov-HTH-Terminator-ptetO-CFP

28.pLac -Rbs(1.0)-lov-tetR-Terminator-ptetO-CFP

29.pLac -Rbs(0.3)-lov- tetR -Terminator-ptetO-CFP

30.pLac -Rbs(0.07)-lov- tetR -Terminator-ptetO-CFP

31.pLac-Rbs(1.0)-lov-HTH-Terminator-ptetO- lux

32.pLac -Rbs(0.3)-lov-HTH-Terminator-ptetO- lux

33.pLac -Rbs(0.07)-lov-HTH-Terminator-ptetO- lux

34.pLac -Rbs(1.0)-lov-tetR-Terminator-ptetO- lux

35.pLac -Rbs(0.3)-lov- tetR -Terminator-ptetO- lux

36.pLac -Rbs(0.07)-lov- tetR -Terminator-ptetO- lux

37.T7 promoter-Rbs(1.0)-lov-HTH-Terminator-ptetO-GFP

38.T7 promoter -Rbs(0.3)-lov-HTH-Terminator-ptetO-GFP

39.T7 promoter -Rbs(0.07)-lov-HTH-Terminator-ptetO-GFP

40.T7 promoter -Rbs(1.0)-lov-tetR-Terminator-ptetO-GFP

41.T7 promoter -Rbs(0.3)-lov- tetR -Terminator-ptetO-GFP

42.T7 promoter -Rbs(0.07)-lov- tetR -Terminator-ptetO-GFP

43.T7 promoter-Rbs(1.0)-lov-HTH-Terminator-ptetO-CFP

44.T7 promoter -Rbs(0.3)-lov-HTH-Terminator-ptetO-CFP

45.T7 promoter -Rbs(0.07)-lov-HTH-Terminator-ptetO-CFP

46.T7 promoter -Rbs(1.0)-lov-tetR-Terminator-ptetO-CFP

47.T7 promoter -Rbs(0.3)-lov- tetR -Terminator-ptetO-CFP

48.T7 promoter -Rbs(0.07)-lov- tetR -Terminator-ptetO-CFP

49.T7 promoter-Rbs(1.0)-lov-HTH-Terminator-ptetO-lux

50.T7 promoter -Rbs(0.3)-lov-HTH-Terminator-ptetO- lux

51.T7 promoter -Rbs(0.07)-lov-HTH-Terminator-ptetO- lux

52.T7 promoter -Rbs(1.0)-lov-tetR-Terminator-ptetO- lux

53.T7 promoter -Rbs(0.3)-lov- tetR -Terminator-ptetO- lux

54.T7 promoter -Rbs(0.07)-lov- tetR -Terminator-ptetO- lux

Transform the parts mentioned in the box into dH5a.

Pick colonies to cultivate. And extract plasmids.

Do all the double digestions and ligations together for the first time. The order is:

1.Promoter + Rbs & Terminator + Reporter

2.Promoter-Rbs + modified protein

3.Promoter-Rbs-modified protein + Terminator-Reporter

Transform fails for several ligation products.

At last, we choose 3 gene circuits for further investigation:

1.araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-GFP

2.T7 promoter -Rbs(0.3)-lov-HTH-Terminator-ptetO-GFP

3.araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO-GFP

4.araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-lux

5.T7 promoter -Rbs(0.3)-lov-HTH-Terminator-ptetO-lux

6.araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO-lux

We run a SDS-PAGE gel to confirm the expression of the modified protein. The result is positive.

In order to verify the modified protein’s characteristic, Juntao Mai does the western-blot. The result is partly positive. Under equal quantity induction of arabinose (0.3%), samples under 450nm wavelength light shows a 30% GFP decay compared with samples incubated in the dark.

We detected the lov-HTH, whose HTH part is modified, by reversed transcription PCR. We‘ve done a series of RT PCR with samples induced by 0.3% arabinose during their incubation and samples incubated without arabinose at the first place. In order to get rid of the effect of light, we incubated all the cells in the dark. We can easily find lov-HTH (modified) mRNA in samples induced by arabinose.

We investigated the time course of the transcription of the arabinose-induced GFP using quantitative real-time PCR. We combined different conditions together and did a series of QRT PCR. We can get the detail in this table.

We decide to use EMSA (Electrophoretic Mobility Shift Assay) to verify if the modified protein can be efficient binding the specific part in ptetO.

Considering the time, cost and the possibility, we give up EMSA.

With propose of testing the protein’s repression ability, we do a fluorescence intensity test. Under an arabinose induction gradient between 0 to 0.3%, we investigate the time course of the two groups of cells’ GFP fluorescence intensity and the absorbance of OD600 at the same time. One group of them is incubated in the 470nm wavelength light and the other one was incubated in the dark. The two curves of RFU per OD600 present 30% differences which is positive.

With one of our classmates’ help, Zining Hou make a wood box for shooting pictures in the dark.

We make our first double layer disk to shoot.

Our instructor helps us make a single colony mathematic model.

We get a series of photos which show a positive result. (araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-lux)

We compare araBAD-Rbs(1.0)-lov-HTH-Terminator-ptetO-lux, araBAD-Rbs(1.0)-lov-tetR-Terminator-ptetO-lux and T7 promoter-Rbs(0.3)-lov-HTH-Terminator-ptetO-lux by putting four double layer disks together (we use ptetO-lux as control). Unfortunately, the light emitted from different disks interfere each other. So we couldn’t get a usable result. But good news is that we assure the light emitted by lux is strong enough to be a signal.

Organize our first association recruiting with Fudan_D, our cooperating team.

Build the wiki