Part:BBa_K2391001

Zinc-responsive Activator Protein (ZAP1)

ZAP1 is a zinc regulated transcription factor that has seven zinc finger domains. It has the ability to, in low concentrations of zinc, repress certain genes. And while in the presence of zinc it binds to certain zinc sensitive promoters inducing transcription.

Usage and Biology

The transcription factor know as ZAP1 is found in Saccharomyces cerevisiae is involved in homeostasis of zinc in cells. It is able to do this by controlling zinc metabolism via gene expression. ZAP1 can be found to be active in cells with limited zinc. And it is repressed in cellular environments with abundant zinc. Autoregulation is how ZAP1 is able to regulate its own expression.

Characterization

Steps taken to build the plasmid (pMOD-ZAP1-GFP-CYC1) 1) After planning through what is needed in the backbone of the final plasmid, we have amplified an integrating plasmid obtained from the Seelig lab in University of Washington. This backbone contains a PBR322 origin of replication, an Ampicillin resistance gene, and a tryptophan marker along with a CYC1 terminator for yeast.

2) For the insert (ZAP1-GFP), we first transformed plasmid obtained from the 2017 DNA Distribution Kit #1, Part:BBa_K895006. This biobrick contains the GFP Open Reading Frame and was made by the Dundee team in 2012. With specific restriction enzymes present in the biobrick prefix and suffix, we digested the GFP-ORF biobrick and this biobrick (ZAP1 promoter) and did a three piece ligation of ZAP1, GFP-ORF and the backbone of PSB1C3.

3) After a successful transformation of the three piece ligation, the team then designed primers that amplifies the backbone of the final plasmid with 20-40 bp overhang sequences that is homologous the insert that is currently in PSB1C3 to do a Gibson assembly to form the final plasmid.

4) We have successful transformation of the final plasmid as seen in the picture (below).

Next steps for Characterization:

1) Transform miniprepped plasmid into competent yeast cells and measure intensity of GFP expression using plate reader.

2) We have planned a rough characterization protocol that is on Washington Team's wiki page under the section 'BioBricks'.

Sequence

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]