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.coli (BL21(DE3)) with a p.Cons-asPink plasmid (BBa_K3182100). The promotor is constitutive which will express AsPink and makes the colonies pink. This can be used to determine whether a 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 microbial cellulose bandage was suspended into 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 specific for the cellulose bandage.]

Figure 2. To test the release mechanism of CBD-asPink, human thrombin and cleavage buffer was added to the bandage and incubated for 16 hours on an end to end rotator. The figure is the result after the incubation with a negative control with only cleavage buffer (left) and human thrombin and cleavage buffer (right)

CBD-asPink with thrombin cleavage
After the washes, human thrombin and cleavage buffer was added to the bandage with bound CBD-asPink to test the release mechanism of the fusion protein. The bandage was then incubated with the solution for 16 hours on an end to end rotator together with an negative control containing cellulose bandage and only cleavage buffer. After the incubation, the supernatant containing thrombin was pink while the negative control containing only cleavage buffer was transparent with a clear pink cellulose bandage (see Figure 2). This indicates that the release mechanism work and the AsPink protein has successfully been released from the bandage into the supernatant.

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