Part:BBa_K3182100

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]

Introduction

pT7-CBDcipA-pCons-AsPink BBa K3182000

Theoretical usage of this part

This part utilizes a pCons-AsPink dropout enabling colour-screening for positive colonies. Using BamHI and PstI or SpeI on both this part assembled in pSB1C3 (or vector of choice) 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.

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".

Examples of biobricks assembled with this method

This method was used to assemble: BBa_K3182103, BBa_K3182104, BBa_K3182105, BBa_K3182106, BBa_K3182107, BBa_K3182108 and all these parts were assembled in a modified version of pUC19 as well, using this method.

Usage and Biology

Design any gene which you want to be fused to the CBDcipA with a BamHI recognition sequence in the 5'-end. The biobrick suffix can be used in the 3'-end. Cut the vector and insert with BamHI and PstI (SpeI also works), remove enzymes and mix, no need for gel purification. Using very high molar ratios might not yield any pink colonies at all, a molar ratio insert to vector of 7-20:1 will yield some pink colonies. Transform the host (BL21 (DE3) for quickest results) and incubate at 37 degrees Celsius overnight, if the color is weak or can't be seen, incubate in 24-37 degrees for an additional 16-24 hours.

Figure 2. E. coli (BL21) cells used for pink-white screening, the cells were incubated for 16 hours in 37 degrees Celsius. BBa_K3182100 was cut with BamHI and PstI to remove pCons-AsPink and BBa_K3182006 (magainin 2) and BBa_K3182104 (CHAP) was ligated into the plasmid. The white colonies indicate a successful ligation. All the colonies that were later colony screened with PCR amplification of the insert and the ampified strand was run on an agarose gel. The gel implied that all screened colonies was successful, i.e. contained BBa_K3182100 with magainin 2 / CHAP instead of pCons-AsPink.

INSERT AGAROSE GEL HERE