Team:Tianjin/Note/6803

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Notes For Modified Cyanobacteria：A Controllable Lipid Producer

Week1(7/31/2016-8/6/2016)

  Jul.31th

  • Cultivation: 30µL mother liquid of Synechococcus sp PCC 7942 (wild type)were cultivated in 10 mL BG-11-media at 30°C and 140rpm.
  • The genome DNA of 7942 was extracted using a Genomic DNA Isolation Kit.

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    Week2(8/14/2016-8/20/2016)

    Synechococcus sp PCC 7942 had no signals of life.

    Igem-6803-week2
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    Week3(8/28/2016-9/3/2016)

      Aug.29th

  • pMV-G19 and pMV-G15 containing our target genes were received.

    • Igem-6803-week3-1
  • Colonies were used to inoculate overnight cultures.

    •   Aug.30th

  • 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.

    •   Aug.31th

  • 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.

    •   Sep.1th

  • 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.

    •   Sep.2th

  • 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.
    • Igem-6803-week3-2
  • Dephosphorylated plasmid and phosphorylated gene 15 were connected.
  • Ligation product was transformed into E.coli via heat shock.

    •   Sep.3th

  • 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.

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    Week4(9/4/2016-9/10/2016)

      Sep.4th

  • 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.

    •   Sep.6th

    Colonies containing gene 13 were used to inoculate overnight cultures.

      Sep.7th

  • 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.

    •   Sep.8th

    13 gene fragment was phosphorylated.

      Sep.9th

  • 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.

    •   Sep.10th

  • 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.
    Igem-6803-week4

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    Week5(9/11/2016-9/17/2016)

      Sep.11th

    Two kinds of plasmids were isolated using a miniprep kit.

      Sep.12th

    Two kinds of plasmids were isolated using a miniprep kit.

      Sep.14th

    The sequencing results for both of them were error.

      Sep.15th

  • 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.

    •   Sep.16th

  • Plasmids pCPC-3031-Ni were isolated using a miniprep kit.

    •   Sep.17th

  • Several PCRs were performed to check if the gene fragments were ligated correctly.
    •   13_fwd and 13_rev on pT-13-19-15
      19_fwd and 19_rev on pT-13-19-15
      15_fwd and 15_rev on pT-13-19-15
      13_fwd and 19_rev on pT-13-19-15
      19_fwd and 15_rev on pT-13-19-15
      13_ wd and 15_rev on pT-13-19-15
    The fourth and sixth ones were not successful.
    Igem-6803-week5
  • Repetition: Phosphorylated 13 was ligated into pT-19-15 and transformed into E.coli via heat shock.

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    Week6(9/18/2016-9/24/2016)

      Sep.18th

    Repetition: Several PCRs were performed to check if the gene fragments were ligated correctly.
      13_fwd and 13_rev on pT-13-19-15
      13_fwd and 19_rev on pT-13-19-15
    The second one was failed.

      Sep.21th

  • Medium preparation :BG-11.
  • 19_ fwd and 15_rev were used to amplify 19-15.

    •   Sep.22th

  • 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.
    • Igem-6803-week6-1
  • The fragment 13-19-15 was ligated onto T vector and transformed into E.coli via heat shock.
    • Igem-6803-week6-2

      Sep.23th

  • 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.

    •   Sep.24th

  • Plasmids pT-13-19-15 were isolated using a miniprep kit.

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    Week7(9/25/2016-10/1/2016)

      Sep.25th

  • 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.
    • Igem-6803-week6-3

      Sep.26th

  • 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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    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


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