Cultivation: 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℃.
We add a single 3`-adenine overhang to each end of the fragment of 19 and then purified with DNA Purification Kit.
19 was ligated into T vectors via TA clone and transformed into E.coli via heat shock.
A colony PCR was performed with five colonies.
Gel electrophoresis showed that 5 colonies were positive for insertion of 19.
Two of these colonies containing 19 were used to inoculate overnight cultures.
15 gene fragment was phosphorylated.
Plasmids containing T vector with 19(pT-19)were isolated using a miniprep kit.
Mono-restriction digest of pT-19 with stu I.
The enzyme-digested product was dephosphorylation.
Dephosphorylated plasmid and phosphorylated gene 15 were connected.
Ligation product was transformed into E.coli via heat shock.
A colony PCR was performed with twelve colonies.
Gel electrophoresis showed that 2 colonies were positive for connecting the plasmid and gene fragment.
These two colonies were used to inoculate overnight cultures.
Plasmids with correct sequence of 19-15 were isolated using a miniprep kit.
Mono-restriction digest of pT-19-15 with Nru I.
The enzyme-digested product was dephosphorylation.
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.
Notice: This page is currently under construction. Contents in this page are temporaory and will be modified several times before the final release. — 2016 iGEM Team Tianjin
