09/09/2014

Today we made the sequencing reaction of our bio bricks (BB_Essential, BB_Bioremediation, BB_Essential + E0840 and BB_Essential + K346004, BB_Essencial + BB_Bioremediation).

We also continued the total DNA extraction from wild bacteria that present resistance to high Hg concentrations using the phenol-chlorophorm method, started on September 6th. And we finally could finish it!! \o/ Will we be able to check on the electrophoretic profile a plasmid presence?!?! Let’s check!

Electrophoretic profile from total DNA extraction from wild bacteria resistant to high Hg concentrations.

1: Total DNA of sample A1* collected from Negro River

2: Total DNA of sample A2* collected from Negro River

3: Total DNA of sample E1* collected from stream 40

4: Total DNA of sample E2 collected from stream 40

5: Total DNA of sample P1* collected from stream 40

6: Total DNA of sample P2 collected from stream 40

7: Total DNA of sample P3 collected from stream 40

8: Total DNA of sample S1* collected from stream 40

*Resistant to 100ug/ml Hg.

We could see that some bacteria present plasmids! Super cool! Next step is to amplify the 16S rRNA and sequence them to identify!

We also continued the new constructions. Today we made plasmid DNA extraction from DH5α with bio bricks K611025 (promoter 1), J23100 (promoter 3) and K516030 (mRFP generator).

Electrophoretic profile from plasmid DNA extraction:

1– 2: Promoter 1 (K611025) in PSB1C3 extracted from DH5α.

3– 4: Promoter 3 (J23100) in J61002 extracted from DH5α.

5– 6: mRFP generator (K516030) in PSB1C3 extracted from DH5α.

We now have plasmids for the new constructions! We need to clone mRFP in JM110 to cut with the restriction enzyme Xbal. For more information, see methods section.