Part:BBa_K1033933:Experience

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2019 iGEM team Linkoping Sweden

2019 iGEM team Linkoping Sweden validated this part.



To visualize the absorbance of AsPink on a petri dish, BL21 (DE3) colonies containing the pUC19-pCons-AsPink part (BBa_K3182100), which was used in the pink-white screening, were incubated for 16 hours in 37 °C. The pCons-AsPink construct was removed and (Magainin 2) and BBa_K3182104 (CHAP) was inserted into the vector. The pink colonies contain AsPink and indicate a religated pCons-AsPink. The white colonies indicate a successful ligation. Below (Figure 1) is a picture of the pink-white screening.

Figure 2. A: E. coli BL21 cells expressing the biobrick, incubated for 16 hours at 16°C at 80 rpm in 1L of LB-miller. B: Lysated (via sonication) BL21s which are expressing the biobrick. It is lysate from the culture above in figure1. Incubated for 16 hours at 16°C at 80 rpm in 1 litre of LB-miller. C:Centrifuged lysate of BL21 culture which express the biobrick. It is the same culture as figure 4 and 5. A pellet of non-lysated bacteria can be observed.

Figure 1. BL21 (DE3) bacteria used for pink-white screening. The white colonies indicate a successful ligation and the pink colonies is AsPink expressing bacteria which signals a false-positive ligation.

CBD-AsPink (K3182000) were expressed in BL21 (DE3) Gold 16 hours at 16°C at 80 rpm in 1L of LB-miller in Figure 1 above. Colonies containing a CBD-pCons-asPink part (BBa_K3182100) is shown above in Figure 2. The construct was used to determine whether a ligation was successful or not. The pink colonies indicate a false-positive ligation, since the pCons-AsPink part had religated. Below is a picture (Figure 2) of the pink-white screening.



CBD-asPink bindning capacity

Figure 3. Lysate (via sonication) from E. coli BL21 was incubated with Epiprotect (microbial cellulose bandage) for 1h and washed thrice with 70% ethanol.

To test the bindning capacity of CBD-asPink (BBa_K3182000) the microbial cellulose bandage was suspended into sonicated lysate of E.coli BL21(DE3) with the expressed fusion protein (Figure 3) 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 4. 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 (Figure 4). 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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