Team:BIT-China/Notebook
- Week 1
- Week 2
- Week 3
- Week 4
- Week 5
- Week 6
- Week 7
- Week 8
- Week 9
- Week 10
- Week 11
- Week 12
- Week 13
- Week 14
- Week 15
- Week 16
- Week 17
- Week 18
- Week 19
- Week 20
- Week 21
- Week 22
- Week 23
We started our project in March, and we have three parts of experiment, basic circuits part, regulation part and Error-prone PCR part. Basic circuits part was finished by Zongzhi Lv's group (including Hui Luo) and Wenxin Bai's group (including Changxin Zhang and Jiaqi Xu), Regulation part was finished by Jinlin Li's group (including Jiadong Zhong and Pengcheng Zhang). Error-prone PCR was finished by Jing Yang's group(including Chengyi Li, Mingming Cao, Xiao Peng and Zeyan Li). The experiments of Regulation part consist of the construction and verification of two pH-induce promoters (P-atp2 and P-asr) and recombinase system. The main work of error-prone PCR (EP-PCR) gruop is to select different promoters by error-prone PCR. The duty of Basic circuits group is to construct our basic resistance sub-system and basic regulation system.
Week1 (Mar 16 -Mar 22)
We extracted the genome of Escherichia coli K12 strain and got the gene LdhA through PCR.
We have successfully got the genome of E.coli K-12 DH5α, preparing for the later use of cloning functional genes.
We have successfully got the functional gene glsA from the genome of E.coli K-12 DH5α by PCR, which encodes an acid-activated glutaminase.
Through digestion, ligation, transformation and cPCR, we have successfully selected the expected strain with glsA. The plasmid is pSB1A3.
Week2 (Mar 23 -Mar 29)
We constructed the gene LdhA to the vector pET28a(we use restriction enzyme cutting sites EcoRI and XhoI), and transformed it into the BMTOP10.
We have successfully got the acid-induced promoter J100071 from the genome of E.coli K-12 DH5α.
Through digestion, ligation, transformation and cPCR, we have successfully selected the expected strain with J100071. The plasmid is pSB1A3.
Week3 (Mar 30 -April 5)
We sequenced the gene LdhA and the result is right. Then we transform the plasmid LdhA-pET28a into the BL21(DE3) and verified it firstly. The result of verification is success.
Through PCR, we constructed the device J23119+B0034+glsA(JBG) for further testing. The plasmid has been replaced by pSB1C3.
Through 3A assembly, we constructed the device J100071+B0034+E1010(RFP) for further testing. The plasmid has been replaced by pSB1C3.
Week4 (Apr 6 -April 12)
We used same method to got and constructed other acid production gene, such as PflB(formic acid production), Pta and AckA (acetic acid production) and so on. At the same time, we verified the gene LdhA again.
Comparing to the strain with the plasmid carrying J23119, we tested the acid resistance ability of the strain with the plasmid carrying JBG.
We set a pH gradient pH=3, 4, 5, 6, 7 and measured the OD600.
However, the result is not ideal.
Week5 (Apr 13 -April 19)
We finished the verification of LdhA(lactic acid production), PflB(formic acid production), Pta and AckA(acetic acid production) three times.
We measured the intensity of red florescence to test the function of J10071 promoter.
Week6 (Apr 20 -April 26)
We finished the verification of DhaB(3-hydroxypropionic acid production), PhaC, PhaA and PhaB(3-hydroxybutyric acid production) three times.
We constructed the standard part with the functional gene glsA. The plasmid is pSB1C3.
We constructed the standard part with the acid-induced promoter J10071. The plasmid is pSB1C3.
Week7 (Apr 27 -May 3)
We constructed all of acid production genes to the pSB1C3, as standard parts.
We did the second cell culture to test the function of glsA, but again, we didn’t get the expected results.
Comparing to the strain with the plasmid carrying J23119, we measured the intensity of proteins expressed in the strain with the plasmid carrying JBG.
Week8 (May 4 -May 10)
We looked around many articles and decided to use P-atp2 and P-asr as pH-induced promoters. At the same time, we extracted the genome of Corynebacterium glutamicum ATCC 13032 strain.
We did the second cell culture to test the function of glsA, but again, we didn’t get the expected results.
Comparing to the strain with the plasmid carrying J23119, we measured the intensity of proteins expressed in the strain with the plasmid carrying JBG.
Week9 (May 11 -May 17)
We obtained the sequence of P-atp2 and P-asr by articles. We tried to constructed P-atp2 to pSB1C3, but there is an EcoRI site exists in P-atp2. So we chose another vector pXMJ19 to construct it.
We did the third cell culture to test the function of glsA, but again, we didn’t get the expected results. We have improved our experimental method, but there are still a lot of difficulties. It’s hard to realize the synchronous culture between the two strain because the different activities of preserved bacteria.
Week10 (May 18 -May 24)
We got P-atp2(Corynebacterium glutamicum ATCC 13032) and P-asr(Escherichia coli K12) through PCR. And linked them to pXMJ19 and pSB1C3 respectively. Then transformed them into BMTOP10.
Week11 (May 25 -May 31)
We used LacZ alpha as a report gene and we verified the strength of two promoters under the different pH environment. The result is success.
Week12 (Jun 1 -Jun 7)
We designed recombinase system to fine-regulation. Meanwhile, we wanted to build a library of pH-induced promoters by error-prone PCR. So we divided to two groups to finish these works. And this week, we searched and looked around the articles to find suitable recombinase and fit condition of EP-PCR.
Comparing to the strain with the plasmid carrying J23119, we measured the intensity of proteins expressed in the strain with the plasmid carrying JBG again.
Week13 (Jun 8 -Jun 14)
This week, we confirmed the recombinase(Cre, Flp, Bxb1 and FimE) we used in our gene circuits and the condition of EP-PCR.
Week14 (Jun 15 -Jun 21)
We extracted the genome of Saccharomyces cerevisiae and transformed mRFP, GFP, K137007(FimE), K907000(Bxb1 gp35) and K907001(Bxb1 gp47) from iGEM kit plates into BMTOP10. Simultaneously, we did the mutation of P-atp2 firstly and selected 157 positive bacteria from more than 500 bacteria
Week15 (Jun 22 -Jun 28)
This week, we had final examination. We didn't have enough time to do any experiments. So we searched and looked around large amount of articles to optimize our experiments.
Week16 (Jun 29 -Jul 5)
We constructed the device K137058(ILL+pTetR+ILR+GFP) according to iGEM and linked it with mRFP. We found a better approach of EP-PCR, so we did again.
We measured the intensity of red florescence at different pH to test the function of acid-induced promoter J100071 again.
Week17 (Jul 6 -Jul 12)
We constructed a new part P-atp2+B0034+K137007(FimE) by over-lap extension PCR(OE-PCR) and transformed it into BMTOP10. In this week, we did amounts of EP-PCR, and were ready to select them next week.
Through PCR, digestion, ligation, transformation and cPCR, we have successfully constructed the standard part glsA on pSB1C3.
Week18 (Jul 13 -Jul 19)
We constructed a new part P-atp2+B0034+K907000(Bxb1 gp35)+K907001(Bxb1 gp47) by OE-PCR and transformed it into BMTOP10. And we started to selected the positive bacteria from our transformant.
We did the fourth cell culture to increase the experimental data and analyzed the common features among the four experiments. However, we didn’t get the expected results again. But we have learned some lessons.
Week19 (Jul 20 -Jul 26)
This week, most members of our two groups attended the iGEM conference held by National Chiao Tung University. So the plenty of members selected others bacteria together.
We added a terminator downstream the gene glsA. Through 3A assembly, we have successfully constructed the complete standard part. Through the result of sequence measuring, it turns out to be completely right.
Week20 (Jul 27 -Aug 2)
We artificially synthesized attP and attB, the recognized sites of Bxb1, by Genwiz, so that we should wait for them. And we totally selected 1082 positive bacteria. We used LacZ alpha as a report gene and started the verification of them.
We have improved our experiment methods and carried out the fifth cell culture. Considering the pH might change after cell culture, we added buffer to control the pH. Moreover, we tried to realize the the synchronous culture by choosing the single bacteria on the plate. Unluckily, the results didn’t meet our requirements.
Week21 (Aug 3 -Aug 9)
This week, we got a measure that we helped Beijing Normal University(BNU) to finish their gene circuit. So we transformed the gene I15008(ho1) and I15009(PcyA) from iGEM kit plates into BMTOP10. And we still did the verification of our mutants.
We measured the intensity of proteins again. It showed that the expression of glsA is critically low. So, we planned to change a strong promoter T7.
Week22 (Aug 10 -Aug 16)
We got the BNU's part (J23100+B0034+PcyA+B0034+ho1+B0015), and constructed it to the pSB1C3. We still verified the mutants this week.
Referring to the paper we read, we did a small experiment to measure the proportion of living cells under extreme pH 3.0, both the testing group and the comparison group didn’t grow well. The difference is not evident.
Week23 (Aug 17 -Aug 23)
We finished the gene circuits of BNU and constructed the new device P-atp2+B0034+K137007(FimE)+B0015+mRFP+ILL+pTetR+ILR+B0034+GFP+B0015 to pSB1C3. We sorted part of our verification data and we were ready to verify the optimize mutants.
Through OE-PCR, digestion, ligation, transformation and cPCR, we planned to ligate p-atp2+B0034+LdhA. However, we cannot select the right one. After analysis, there were some problems with our plasmid, so we replace it and plan to try again.