Team:Tianjin/Note/6803

Notes For Cyanobacteria

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.

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Week3(8/28/2016-9/3/2016)

  Aug.29th

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

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.

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.

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

  Sep.11th

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.

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.

The fragment 13-19-15 was ligated onto T vector and transformed into E.coli via heat shock.

  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.

  Sep.26th

Plasmids pT-13-19-15 were handed in for sequencing, which confirmed its correctness.

The sequencing results for them were correct.

  Sep.28th

We transformed the expression vector, pCPC-3031-Ni-13-19-15, into Synechocystis 6803 via electroporation.

The story of 6803 is not over. Other team members will show the completed process during the presentation.
I will cherish the time spending with you for my whole life. ———by Jiawei Chen and Liangyu Qian

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clear 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