Team:Peking S/lab/notebook/qx

In our project, my job is mainly to construct the competitor module, as well as characterize its performance. I also have a partner, Chen Yiwei, to fulfill this task together.

July

7.1 - 7.7

We did four rounds of PCR to get the ptsG1+gfp. The gfp here refers to BBa_E0840 from the iGEM 2011 Parts Kit. In each step, we added around 30bp of the ptsG1’s 5’ untranslated region (5’ UTR) to the former product, and we finally got the construct for the fused protein. We sequenced the plasmids and it turned out to be correct.

7.8 - 7.14

We started to construct the SgrS(wt) module. To begin with, we managed to get the sequence from the k12 strain of E. coli. Then we added the constitutive promoter BBa_J23106 and the terminator BBa_B0015 to flank the SgrS. The product here was “Pc+SgrS(wt)+Terminator”. We also substituted the constitutive promoter with the arabinose-inducible promoter PBAD (BBa_I13453) and got “PBAD+SgrS(wt)+Terminator”.

7.15 - 7.21

According to the same protocol, we tried to construct the remaining ptsG mutants, i.e. ptsG1’, ptsG2, and ptsG2’. Since the primers were designed in the same manner as ptsG1, we expected to go through this part of molecular cloning smoothly. However, things got stuck here. The PCR always aborted half the way, and all we could do is to slightly change the reaction system and repeat the procedure over and over again. Finally, it seemed we had got all four candidates for further characterization. To our great surprise, the sequences proved to be all the same: they were all identical to ptsG1. Then we found out that the problem emerged from the design of the primers. We had four sequential forward primers, and the ignorance of the overlapping part of them led to the reverse mutation that compensated the adjustment we did to ptsG1’, 2, and 2’ from ptsG1.

7.22 - 8.7

These days we mainly focused on executing site mutation PCR to get the construction SgrS1 and SgrS2. We met another great problem here. Though we conducted our experiment exactly according to the protocol, the PCR always failed. All the products remained in the holes of the gel after electrophoresis, and further experiments thus couldn’t be done. Again we had to repeat the experiment to figure out what was the optimal condition to get things done. By troubleshooting, we thought problems lied in the specificity of our primers and the fidelity of the DNA polymerase. By changing our template as well as the enzyme we used, after days of tedious work, we made it. SgrS1&2 were done.

August

8.1

8.2

8.4

8.5

8.6

8.7

8.8

8.9

8.10

8.11

8.12

8.13

8.14

8.15

8.16

8.17

8.18

Transform of luxR(1-8O, 936bp, I0462), plux'(1-14P, 30bp, R0061), luxI(3-14A, 711bp, K081015)

8.19

Retransform of plux', luxI; pc+luxR

8.20

Ligation of plux'+T7ptag(2673 bp)+terminator, pT7 (I719005) +luxI.

8.21

CAI-1 induce of the CAI-1 system.

8.22

Ligation of pc+luxR, transform of luxI(1-14C, 661bp, C0261)

8.23

pT7+luxI 1,3,5 sequencing right. Digestion of luxI(1-14C)

8.24

Ligation of pT7+luxI(no terminator), (pBAD+supD)+(plux'+T7ptag (6))+4K5.

8.25

pc+luxR(1-8O) was wrong in part! Transform of luxR(2-4O,799bp,J37033)

8.26

CAI-1 induce of the CAI-1 system.

8.27

pT7+luxI(no term). No. 1, 3 sequencing right. Ligation of pT7+luxI + GFP(ssrA tag) for sequencing. Colony PCR, (1) 1,3,4,5 (3)1,2,3,4. PCR for 1-4O, 1-8O(in 2009 Distribution). Digest 1-2M(RBS). Transform of pc(J23100, 1-18C), luxR(1-4O).

8.28

Ligation of pc+1-8O. (pBAD+supD)+(plux'+T7ptag (6))+4K5 sequencing wrong! Digest (pBAD+supD).

8.29

pT7+luxI+GFP(ssrA tag) sequencing wrong (No luxI?!) Ligation again for 3A (psb1C3). Ligation for RBS+luxR(1-4O). Ligation (pBAD+supD)+(plux'+T7ptag (6))+4K5 again. (btw T7ptag sequencing right).

8.30

(pBAD+supD)+(plux'+T7ptag (6))+4K5 16, 32 for sequencing. pT7+luxI+GFP(ssrA tag) colony PCR: (1). 7, 8, 10; (3). 1, 3, 4, 5, 6, 7, 9, 10. Digest RBS+luxR(1-4O).

8.31

pT7+luxI+GFP(ssrA tag) Digest check: (1). 7, 8, 10; (3). 4, 6, 7, 9. for sequencing. Ligation of pC+RBS+luxR send for sequencing.

September

[TOP]

9.1

RBS+luxR sequencing right in the end! Religation of (pBAD+supD)+(plux'+T7ptag (6))+4K5 and (pBAD+supD)+(plux'+T7ptag (6))+3C5, doubt whether (pBAD+supD) has been right, redigest.

9.2

pT7+luxI+GFP(ssrA tag) sequencing right. Ligation of (pT7+luxI+GFP)+(pC+RBS+luxR) psB1A3. Religation of (pBAD+supD)+(plux'+T7ptag (6))+4K5 and (pBAD+supD)+(plux'+T7ptag (6))+3C5. (pBAD+supD)+(plux'+T7ptag (6))+4K5 (43) sequencing right!

9.3

(pT7+luxI+GFP)+(pC+RBS+luxR) psB1A3 colony PCR right. Double transform for induce.

9.4

Induce of the feedback circuit.

9.5

Induce of the feedback circuit. (pT7+luxI+GFP)+(pC+RBS+luxR) psB1A3 sent for sequencing.

9.6

9.7

9.8

9.9

9.12

9.13

9.14

9.15

9.16

9.17

9.21

9.22

9.23

9.24

9.25

9.26

9.27

9.28

9.29

9.30

October