Difference between revisions of "Part:BBa K182005"

Revision as of 12:30, 21 August 2009

TetR regulated by CI operator (RBS+, Term-) with LVA tag

This is a part using the biobrick BBa_K182004 as the upstream part and BBa_S03518 as the downstream part.

BBa_K182004 is a cI regulated promoter. The promoter has two binding sites for the cI repressor and is repressed when it binds.It is identical to BBa_R0051 apart from a point mutation in the -35 promoter binding region(15th base is C instead of T).

BBa_S03518 is a TetR protein with a Ribosome Binding site upstream and an LVA tag attached to its end. TetR binds to TetR regulated promoters such as BBa_R0040. aTc (anhydrotetracycline) binds to TetR causing it to be released from the promoter and reintroducing transcription.

This part comes without a terminator.

BBa_K182005

This part was constructed using a copy of BBa_R0051 (Lambda CI Promoter) with a point mutation as the upstream part and BBa_S03518 (RBS + TetR) as the downstream part. Even though this BioBrick was lacking a terminator we were able to test it, by transforming it into E.coli together with BBa_I13600.

BBa_I13600 contains a TetOperator (which is repressed by the Tet Repressor) and controls expression of Cyan Florescent Protein (CFP). Under normal circumstances the BBa_I13600 expresses CFP and this can be visualised under a florescent microscope.

Transforming BBa_K182005 into these cells should stop the production of CFP, if it works as anticipated, due to our Tet repressor binding to the Tet Operator which would stop the expression of CFP.

Since K182005 is on an Ampicillin / Tetracycline vector and I13600 is on an Ampicillin vector, we decided to first transform I13600 into a cell and select for Ampicillin. We then checked for CFP expression and made the cells competent. After this we transformed K182005 into these cells and selected for Tetracycline.

With both plasmids now in the cell we checked for fluorescence as can be seen in the picture below.

As can be seen in the picture we observed no fluorescence, however once we added Anhydrotetracycline we found that CFP was yet again expressed, indicating that the K182005 does in fact express the Tet Repressor protein and that this protein works as expected.

Sequence and Features