Part:BBa_K3970021

pGAP-CpcE-tCYC1

Usage and Biology

CpcE 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-cpcE-up and primer:cpcE-dn to get the fragment of CpcE.

We use primer:cpcE-CYC1-up and primer:CYC1-homo-dn to get the fragment of CYC1.

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

Primers we use to construct cpcE expression cassette:

GAP-cpcE-up (43-mer): Attagatctcgccaccatgccatgttgtcattagaacaattgg

cpcE-dn (20-mer): gaccatcaggaggtcaattg

cpcE-CYC1-up (40-mer): Ttggctcaaagatgatggtccgagtcatgtaattagttat

Xbal-GAP-up: tcgctccccatttctctagtcattatcaatactgccatttcaaagaat

GAP-cpcA-dn: TTCAGTAATCGGTGTTTTCATggtggcgagatctaattcg

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 cpcE.

Experiment results

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]