09/02/2014

Our transformations in JM110 with bio bricks BBa_K516030 (RBS + RFP + TT) and BBa_I712074 (promoter) dind’t grow, we think that this is because the bacteri efficiency is too low. To solve this, we’re going to use the same method as on the first team transformations, two months ago: first cloning in DH5α and after in JM110 to remove the dam methylation from the Xbal restriction site. For more details, please, access Materials and Methods.

Besides the transformtion of DH5α with bio bricks BBa_K516030 and BBa_I712074, we made a new test with mercury discussed yesterday.

We made inoculum in liquid medium LM (LB + Mercury chloride) with concentration of 2ug/ml, 5ug/ml and 10ug/ml the construction and the hosts:

- Only DH5α, without resistence to Hg.

- DH5α with BB_Essential + BB_Bioremediation in PSB1C3

- DH5α with BB_Essential + E0840 in PSB1C3

- DH5α with BB_Essential + K346004 in PSB1C3

We also made inoculum in liquid medium LM 50ug/ml and 100ug/ml 10 colonies from Negro River and the stream contaminated with industry residuals (samples A1, A2, A3, A4; E1, E2; P1, P2, P3; S1) that already showed to be able to grow in medium LM, in high concentrations! We’re being really careful!

In three days we’re gonna have the results. We’re cheering that DH5α with BB_Essential + E0840 in PSB1C3 and DH5α with BB_Essential + K346004 in PSB1C3 grow up in LM!!!!

Today we also started a new journey: Sequencing our Bio bricks. On these first steps we’re going to adjust the methods and the necessary plasmid quantitie for a good sequencing. For that we made a colony PCR and of plasmid, that we showed previously it cotains our bio brick in PSB1C3. (for more details, access previous days). We used the plasmids VF2 and VR.

Colony PCR and plasmids with our bio bricks in PSB1C3.

1 – 2: Colony PCR obtained from DH5α with BB_Essential + E0840

3 – 4: Colony PCR obtained from DH5α with BB_Essential + K346004

5 – 6: Colony PCR obtained from DH5α with BB_Essential + BB_Bioremediation

7: Test for necessary plasmid concentration. BB_Essential in PSB1C3 diluted 1:100

8: Test for necessary plasmid concentration. BB_Essential in PSB1C3 diluted 1:250

9: Test for necessary plasmid concentration. BB_Essential in PSB1C3 diluted 1:500

10: Test for necessary plasmid concentration. BB_Essential + BB_Bioremediation in PSB1C3 diluted 1:100

11: Test for necessary plasmid concentration. BB_Essential + BB_Bioremediation in PSB1C3 diluted 1:250

12: Test for necessary plasmid concentration. BB_Essential + BB_Bioremediation in PSB1C3 diluted 1:500

With this test we can conclude that we’ll need to make lower dilutions or don’t dilute at all our bio bricks to amplify them. We’re going to do more tests! 