Team:Peking S/lab/notebook/cyw

Yiwei Chen's Notebook

July

[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

August

[TOP]

8.1~8.9

8.10

We turned back to our ptsGs. Using ptsG1 as the template, we conducted another series of site mutation experiments to get ptsG1’, ptsG2, and ptsG2’. This time it went much more successfully. After the addition of Pc and PBAD upstream, we could finally undertake the task of characterization of our competitor.

8.11

8.12

8.13

8.14

8.15

8.16

8.17

8.18

Up to now, we had “Pc+ptsG+gfp” series (wt, 1, 1’, 2, 2’), “PBAD +ptsG+gfp” series (as above), “Pc+SgrS+Terminator” series (wt, 1, 2), and “PBAD+SgrS+Terminator” series (as above) in all.

8.19

What remained to be done is to put them on appropriate-copy-numbered plasmids to get our competitor’s best performance. We first tried to put the “PBAD” class on the medium-to-low copy number plasmids pSB3T5, while let the “Pc” class untouched on their high-copy-number plasmids.

8.20

After double transformation, we conducted the pre-experiments to qualitatively evaluate the competitor’s performance. 

8.21

The experimental protocol was as follows. We used 10-2 mol/L arabinose to induce the PBAD, and every 5 experimental groups are coupled with 2 controls, i.e. culture solution without any arabinose in it.

8.22

The result was that whether ptsG or SgrS was under Pc, its property is dominant. That suggested Pc overweighed PBAD completely under such a configuration. A change in copy number was needed.

8.23

What’s more, the ptsG1 and ptsG2 fused gfp performed badly in the experiments; their fluorescence was hardly discernible even under Pc. So we dropped them and chose ptsG1’ and ptsG2’ to go on.

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

By changing backbones, we got “Pc+ptsG+gfp+pSB4K5” series (1’, 2’, wt), and co-transformed them with “PBAD+SgrS+Terminator+pSB1A3” series (1, 2, wt). Then we got 3×3=9 constructs in total.

9.2

A matrix was built up to test the repression capacity of the conjugated ptsG/SgrS systems and the orthogonality of the cross systems. The result was as expected and not bad.

9.3

However, in one of our experiments, we discovered that 10-2 mol/L arabinose alone could repress the expression of GFP, probably by activated the function of endogenesis SgrS(wt) of the E. coli.

9.4

To eliminate the interference of this, we decided to displace PBAD with salicylic acid inducible promoter PSal, since salicylic acid wouldn’t crosstalk with the ptsG/SgrS system.

9.5

Besides, we also had “PBAD+ptsG+gfp+pSB3T5” series (1’, 2’, wt) co-transformed with “Pc+SgrS+Terminator+pSB1A3” series (1, 2, wt). Since the PBAD needed to be replaced, this part of experiments was abandoned.

9.6~9.30

By changing backbones, we got “Pc+ptsG+gfp+pSB4K5” series (1’, 2’, wt), and co-transformed them with “PBAD+SgrS+Terminator+pSB1A3” series (1, 2, wt). Then we got 3×3=9 constructs in total. A matrix was built up to test the repression capacity of the conjugated ptsG/SgrS systems and the orthogonality of the cross systems. The result was as expected and not bad. However, in one of our experiments, we discovered that 10-2 mol/L arabinose alone could repress the expression of GFP, probably by activated the function of endogenesis SgrS(wt) of the E. coli. To eliminate the interference of this, we decided to displace PBAD with salicylic acid inducible promoter PSal, since salicylic acid wouldn’t crosstalk with the ptsG/SgrS system. Besides, we also had “PBAD+ptsG+gfp+pSB3T5” series (1’, 2’, wt) co-transformed with “Pc+SgrS+Terminator+pSB1A3” series (1, 2, wt). Since the PBAD needed to be replaced, this part of experiments was abandoned.

9.30