Part:BBa_K3970023

pGAP-CpcF-tCYC1

Usage and Biology

CpcF encodes phycocyanin alpha Phycocyanobilin lyase which is one part of the Phycocyanobilin lyase catalyzing alpha phycocyanin and 3Z-phycocyanobilin.Phycocyanobilin lyase is required for the chromophorylation of α-phycocyanin to form holo-α-subunit.

In order to get our gene comes from Synechococcus lividus PCC 6715 chromosome, which is a kind of heat-resistant algae. We use other species’s CpcE gene to blast it on NCBI and fortunately we find gene CpcE whose source is PCC 6715.

Construction

We use primer:GAP-cpcF-up and primer:CYC1-dn to get the fragment of CpcE+CYC1.

(For the cpcE sequence is synthesized by the company and as you know the single sequence is not stable so the company suggest us to directly synthesized it on the plasmid. It occurred that there is a CYC1 termination follows the CpcF sequence, so we just have to do one PCR too get two sequence. )

We use primer:homo-GAP-up and primer:GAP-dn to get the fragment of GAP.

Primers we use to construct cpcE expression cassette:

GAP-cpcF-up: attagatctcgccaccatgccTATGGCGGCGCTGCAACTTC

CYC1-dn: ggccgcaaattaaagccttcgag

homo-GAP-up: tcgctccccatttctctagtcattatcaatactgccatttcaaagaat

GAP-dn: ggcatggtggcgagatctaat

Experiment results

The fragments of the constructed plasmid were recovered

M: Marker
1-4: CYC1(terminator)
5-8: cpcE
9: GAP (promoter)
10-13  : cpcF
14-17  :cpcA

The picture shows how we use corresponding primers to get the expression cassette of cpcF.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]