Part:BBa_K3182100

pT7-CBDcipA-pCons-AsPink

AsPink dropout enabling colour-screening for positive colonies. Using BamHI and SpeI or PstI on both this part assembled in pSB1C3 and the insert of choice will yield a fusion protein between CBDcipA and the insert. The fusion protein can later be cleaved with thrombin to yield two separate proteins. The C-terminal fusion will have one glycine and one serine added to the N-terminal of the protein.

The part has a very strong expression with a T7 promotor (BBa_I719005) as well as a 5'-UTR (BBa_K1758100) region which has been shown to further increase expression in E. coli (BBa_K1758106), ([http://www.ncbi.nlm.nih.gov/pubmed/2676996 Olins et al. 1989]), ([http://www.ncbi.nlm.nih.gov/pubmed/23927491 Takahashi et al. 2013]). This requires the T7-RNA-polymerase and bacteria with this should only be used after the C-terminal fusion protein has been assembled.

Figure 1. The first plasmid (left) contains pCons-AsPink which results in pink colonies. When BamHI is used together with either SpeI or PstI pCons-AsPink is cut out (middle plasmid) and replaced with a compatible biobrick such as an antimicrobial agent (right plasmid). The colonies will then be white due to pCons-AsPink being cleaved from the plasmid. This results in a "pink-white screening".

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Usage and Biology

Incubating the bacteria, first in 37 degrees Celsius for 16 hours and then 16 degrees for 24 hours gave a very weak color. A protein production host, such as BL21 (DE3) gave the best results, where color development could be seen after the first incubation.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 592
  Illegal NheI site found at 615
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 580
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]