Part:BBa_K1033933:Experience

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2019 iGEM team Linkoping Sweden

2019 iGEM team Linkoping Sweden validated this part.

Growth of p.cons-asPink
The agar plate contains e.Col (BL21(DE3)) with p.Cons-AsPink (BBa_K3182100) which expresses as-Pink and makes the colonies pink instead of white. These colonies was then later used for color screening to see if the ligation was successful.

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.

Figure 1. Lysate (via sonication) from BL21 E. coli was incubated with Epiprotect (microbial cellulose bandage) for 1h and washed thrice with 70% ethanol.]

CBD-asPink bindning capacity

To test the bindning capacity of CBD-asPink (BBa_K3182000) the white microbial cellulose bandage was into the sonicated lysate of BL21(DE3) with the expressed fusion protein (see Figure 1) and incubated for 30 minutes. The bandage was then washed thrice in 70% ethanol which confirmed that the bindning of CDB-asPink was still intact after the washes to the bandage.

Figure 2. To test the release mechanism, human thrombin and cleavage buffer was added to the bandage and incubated for 16 hours on an end to end rotator.

CBD-asPink thrombin cleavage

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