Week1(8/24/2016-8/30/2016)
We used PCR to amplify the CpxR promoter and RFP gene from plasmid pUC57, and then we recycled the amplified fragment from the agarose gel. Then we use Xba1 and Pst1 enzyme to cut the plasmid pUC19 and CpxR-RFP fragment.
We linked the cut plasmid and CpxR-RFP fragment together and transformed the recombinant plasmid to E.coli.
We used PCR to amplify the PETase gene and then recycled them from the agarose gel.
We cultured the grown-up E.coli which had been transformed into recombinant pUC19 into liquid LB+Amp culture medium overnight.
We isolated the recombinant plasmid from the E.coli cultured last night. Then we use Xba1 and Pst1 enzyme to cut the plasmid to verify the plasmid was successfully constructed. The result was we succeeded.
We cut the recombinant plasmid pUC19 with enzyme EcoR1 and Sac1 and then we recycled it from the agarose gel. We stored the recycled product in -30℃ in order to wait for the PETase gene transformed into it.
In order to verify the inclusion body sensing effects of CpxR promoter, we selected a colony of E.coli with recombinant plasmid pUC19 and cultured them in 37℃ for 6 hours and then rose the temperature to 42℃ and culture it overnight.
The result of the verification experiment last night was unsuccessful for there was only ultralow red fluorescence was detected, which was considered the basic expression of RFP.
We redesigned the experiment and set 3 groups:
1. E.coli with standard RFP gene from our own laboratory.
2. E.coli with our recombinant plasmid pUC19 and we cultured them in 37℃ all through.
3. E.coli with our recombinant plasmid pUC19 and we first cultured them in 37℃ and after 6 hours transferred them to 42℃.
The result was still unsuccessful for the 2nd and 3rd group showed ultralow red fluorescence and only 1st group showed high enough red fluorescence.
We redesigned the experiment again. We decided to transformed the recombinant plasmid pUC19 and pET21A which was from the protein modification group and had PETase gene in it into E.coli at the same time.
We cut the pUC57 with enzyme Xba1 and Pst1 and recycle the skeleton part from the agarose gel.
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We linked the remained cut CpxR-RFP fragment into the skeleton and then transformed the recombinant pUC57 and the pET21A into E.coli at the same time.
The transformation last night turned to be a failure. We tried it again.
The transformation last day seemed to be successful for the colonies were visible in LB+Amp plate. However, we use PCR to verify and it turned out that the fragment had not been linked into the plasmid.
We finally gave up the former design and decided to link the PETase gene into the plasmid pUC19. However, we did not have the key enzyme Sal1 so we started to construct the TPA positive feedback system.
We first prepared the TPA standard solution (5g/L) for further use. Then we use PCR to amplify the TPA-sensing leader sequence, PGK1 promoter, CYC1 terminator, RFP gene, TPA regulation protein gene (tpaR), TPA transporting protein gene (tpaK). Then we cut the fragments above and plasmid pRS413, pRS415, and pYES2 with corresponding enzymes and recycled the fragments from agarose gel.
We linked the fragments together by this way:
1. pYES2-leader-PGK1-RFP.
2. pRS413-PGK1-tpaK-CYC1.
3. pRS415-PGK1-tpaR-CYC1
Then we use PCR to verify the success and all of the plasmids were correctly constructed. Then we transform the there plasmids into Saccharomyces cerevisiae.
The key enzyme Sal1 arrived and we isolate the plasmid pET21A. Then we use BamH1 and Sal1 to cut both plasmid and PETase gene, then linked them together and transformed the recombinant plasmid into E.coli.
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The transformed Saccharomyces cerevisiae had grown to visible colony in Sc-Ura-Leu-His plate. Then we use colony PCR to verify the plasmids had been transformed into the cells. The result is successful so that we streaked more plates.
We cut the T7 promoter-PETase gene-T7 terminator fragment with enzymes EcoR1 and Sac1. Then we linked it to the already cut plasmid pUC19 (cut in August 28th). Then we transformed the recombinant plasmid into E.coli.
We cultured the transformed Saccharomyces cerevisiae into Sc-Ura-Leu-His culture medium in 30℃. We added TPA standard solution in this way:
1. Group 1: did not add TPA.
2. Group 2: add 1000μL TPA standard solution.
3. Group 3: add 100μL TPA standard solution.
4. Group 4: add 10μL TPA standard solution.
5.Group 5: add 1μL TPA standard solution.
We cultured the transformed E.coli into LB+Amp culture medium. Then add 1.5μL IPTG to induce the expression of PETase gene.
We first detect the red fluorescence of E.coli, however, the experiment group had almost no increase of red fluorescence relative to control group. We changed the induction wavelength and scan the whole wavelength of emission, but we did not receive any result we expected.
The TPA positive feedback system seemed to have minor effection for there were a little increment of red fluorescence of the 5th group relative to the 1st one.
We doubt that it might be the RFP in the kit was useless. We isolated the pET21A and used PCR to amplify the RFP gene.
We cut the RFP gene and pET21A gene with enzymes Xba1 and Sac1, then we linked them and transformed it into E.coli.
We cultured the transformed E.coli and added IPTG to induce the expression of RFP, and this time the red fluorescence was clear enough that could be seen directly.
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Week4(9/14/2016-9/20/2016)
Colonies containing gene 13 were used to inoculate overnight cultures.
Plasmids were isolated using a miniprep kit.
Amplification of 13 with Q5 High-Fidelity DNA Polymerase out of E.coli
13 amplification at 65.0°C with 13.rev/fwd primes
PCR worked, positive control worked, no amplification of 13
The fragments of 13 were purified with PCR Purification Kit.
13 gene fragment was phosphorylated.
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Insertion of Ni promoter and ligation of 13-19-15
Ni inducible promoter was ligated into pCPC-3301 vector.
Phosphorylated 13 was ligated into pT-19-15 and transformed into E.coli via heat shock.
Single colonies were obtained by plating.
A colony PCR of Ni inducible promoter(pCPC-3301-Ni) was performed with 12 colonies.
Two of the successful ones were used to inoculate overnight cultures.
A colony PCR of pT-13-19-15 was performed with 7 colonies.
Two of the successful ones were used to inoculate overnight cultures.
Two kinds of plasmids were isolated using a miniprep kit.
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Week5(9/21/2016-9/27/2016)
Plasmids pT-13-19-15 were handed in for sequencing, which confirmed its correctness.
The sequencing results for both of them were error.
Colonies containing Ni inducible promoter were used to inoculate overnight cultures.
PCR was performed to check if the gene fragments were ligated correctly.
13_ fwd and 15_rev on pT-13-19-15
Gel electrophoresis showed that it failed.
Plasmids pCPC-3031-Ni were isolated using a miniprep kit.
Several PCRs were performed to check if the gene fragments were ligated correctly.
1.13_fwd and 13_rev on pT-13-19-15
2.19_fwd and 19_rev on pT-13-19-15
3.15_fwd and 15_rev on pT-13-19-15
4.13_fwd and 19_rev on pT-13-19-15
5.19_fwd and 15_rev on pT-13-19-15
6.13_ wd and 15_rev on pT-13-19-15
The fourth and sixth ones were not successful.
Repetition: Phosphorylated 13 was ligated into pT-19-15 and transformed into E.coli via heat shock.
Repetition: Several PCRs were performed to check if the gene fragments were ligated correctly.
1.13_fwd and 13_rev on pT-13-19-15
2.13_fwd and 19_rev on pT-13-19-15
The second one was failed.
Medium preparation :BG-11.
19_ fwd and 15_rev were used to amplify 19-15.
Gel electrophoresis showed that amplification of fragments was successfull.
Ligated 13 and 19-15 via overlap PCR.
This PCR worked well and the products were extracted from the gel using a Agarose Gel DNA Extration Kit.
Restriction digest on pCPC-3031-Ni with Sac I.
The fragment 13-19-15 was ligated onto T vector and transformed into E.coli via heat shock.
A colony PCR of pT-13-19-15 was performed with 12 colonies.
Two of these colonies containing pT-13-19-15 were used to inoculate overnight cultures.
13-19-15 gene fragment was phosphorylated.
Phosphorylated 13-19-15 was ligated into pCPC-3031-Ni and transformed into E.coli via heat shock.
Plasmids pT-13-19-15 were isolated using a miniprep kit.
A colony PCR of pCPC-3031-Ni-13-19-15 was performed with 12 colonies.
Three colonies were proved to be true. And two of them were used to inoculate overnight cultures.
