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From 2011.igem.org

Contents 1 July 1.1 7.3 1.2 7.4 1.3 7.5 1.4 7.6 1.5 7.7 1.6 7.8 1.7 7.12 1.8 7.13 1.9 7.14 1.10 7.15 1.11 7.16 1.12 7.18 1.13 7.19 1.14 7.20 1.15 7.21 1.16 7.22 1.17 7.23 1.18 7.29 1.19 7.30 1.20 7.31

July

Mon	Tue	Wed	Thu	Fri	Sat	Sun
-	-	-	-	1	2	3
4	5	6	7	8	9	10
11	12	13	14	15	16	17
18	19	20	21	22	23	24
25	26	27	28	29	30	31
-	-	-	-	-	-	-

[TOP]

7.3

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.

7.4

7.5

7.6

7.7

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”.

7.8

Given that we were supposed to insert an RNase E cut site upstream of the SgrS in Cell C constructed by Sun Rui, we also built up “PBAD+RNase E cut site+SgrS(wt)” in case for future use.

7.12

7.13

7.14

7.15

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.

7.16

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.

7.18

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.

7.19

7.20

7.21

7.22

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.

7.23

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.

7.29

7.30

7.31