08/20/2014

Heeello!!! What’s up? Today we solved the molecular question from yesterday, of the vector that wasn’t releasing our bio brick! And the solution is: the BB_Essential + BB_Bioremediation fragment has 3.057 base pairs, and the vector pBSK has 2.861, that’s about 196 base pairs difference! The electrophoretic profile shows only one big band in 3.000 base pairs (comparing it to the marker). Hour hypothesis is that both are there, but we couldn’t separate it! The same hypothesis applies for BB_Essential + E0840! The vector PSB1C3 has 2.070 base pairs and the fragment around 2.136, that’s 66 base pairs difference! The profile shows just one big band at 2000 base pairs! Our construction worked, that’s for sure! To prove that they’re in the right size we developed the following strategy:

We’re going to digest the vectors (PBSK and PSB1C3) with our bio bricks to linearize (using EcoRI), and then digest to release the inserted gene (with EcoRI and PstI). The linear band should present double of band’s size cut to separate vector from fragment. That means, the fragment and BB_Essential + BB_Bioremediation vector cut shows around 3.000 base pairs, the vector containing the linear fragment should show 6.000! Isn’t obvious?

To demonstrate our hypothesis, we used the following system for digestion:

The digestion took 1 hour and a half at 37°C.

Electrophoretic profile of enzymatic digestion:

1- BB_Essential + K346004 not digested.

2- BB_Essential + K346004 cut with EcoRI.

3- BB_Essential + K346004 digested with EcoRI and PstI.

4- BB_Essential + E0840 not digested.

5- BB_Essential + E0840 cut with EcoRI.

6- BB_Essential + E0840 digested with EcoRI and PstI.

7- BB_Essential + BB_Bioremediation not digested.

8- BB_Essential + BB_Bioremediation cut with EcoRI.

9- BB_Essential + BB_Bioremediation digested with EcoRI and PstI.

Woohoo!!! We proved our hypothesis!!! Tomorrow we have one more tricky question to solve: How we’re going to separate the vector and the fragment?