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.

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

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.

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.

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.

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.

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.

  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.

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.

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.

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

The sequencing results for them were correct.

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

The story of 6803 is not over. Other members will show the completed process during the presentation. I will cherish the time for a lifetime.