Team:Tianjin/Note/R-R

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Notes--R-R system

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.
    • Show More IMG_2956

    Week2(8/31/2016-9/6/2016)

  • 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.
    • Show More IMG_2956

    Week3(9/7/2016-9/13/2016)

  • pMV-G19
  • pMV-G15
  • Colonies were used to inoculate overnight cultures.
  • Plasmids were isolated using a miniprep kit.
    • Amplification of 19 and 15 with Q5 High-Fidelity DNA Polymerase out of E.coli
  • 19 amplification at 65.0°C with 19.rev/fwd primes.
    • PCR worked, positive control worked, no amplification of 19.
  • 15 amplification at 65.0°C with 15.rev/fwd primes.
    • PCR worked, positive control worked, no amplification of 15.
  • A product length of 900 bp was expected. The gel shows that most plasmids seem to be correct.
  • This result was confirmed by sequencing.
  • The fragments of 19 and 15 were purified with PCR Purification Kit.
    • Show More Ligation of 15 with 19
  • 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.
    • Show More IMG_2956

    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.
    • Show More 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.
    • Show More

    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.

    Week6(9/28/2016-10/2/2016)

  • 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.

    Week7(9/26/2016-10/2/2016)

  • Plasmids pT-13-19-15 were handed in for sequencing, which confirmed its correctness.
    • The sequencing results for them were correct.

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