08/21/2014

Hi!!! Remember we had one more question to solve today? How are we going to separate our biobrick from the vector (BB_Essential + BB_Bioremediation) for cloning it in PSB1C3, considering that it has around 3.000 base pairs and the vector PBSK also has around 3.000 base pairs! We solved it and developed the following strategy:

Super cool, isn’t it?! Beside cloning our linked bio bricks (BB_Essential + BB_Bioremediation), we also will clone it separately and send it to Registry on PSB1C3! To clone separated bio bricks, we don’t need to use the previously mentioned strategy, we will purify the fragment from the agarose gel and bind it to PSB1C3 after! So, let’s start! We used the following system:

Electrophoretic profile of digestion:

1 : BB_Essential + BB_Bioremediation in PBSK not digested

2: BB_Essential + BB_Bioremediation in PBSK digested with EcoRI + PstI

3: BB_Bioremediation in PBSK not digested

4: BB_Bioremediation in PBSK digested with EcoRI + PstI

5 : BB_Essential in PBSK not digested

6: BB_Essential in PBSK digested with EcoRI + PstI

7: BBa_J04450 in PSB1C3 not digested

8: BBa_J04450 in PSB1C3 digested with EcoRI + PstI.

We took 10ul of samples 2 and 8, then let it overnight.

The remaining samples were purified to use in a binding system later.

Electrophoretic profile from purified samples

1: BB_Bioremediation in PBSK cut with EcoRI and PstI

1: BB_Bioremediation + BB_Essential in PBSK linearizado.

2: PSB1C3 linear.

3: BB_Essential in linear PBSK.