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Team:Aberdeen Scotland/WetLab/lacilatch/results
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The second BioBrick constructed as part of the LacI latch module was 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. | The second BioBrick constructed as part of the LacI latch module was 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. | ||
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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. | 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. | ||
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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. | 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. | ||
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| - | Since K182005 is on an Ampicillin / | + | Since K182005 is on an Ampicillin / Chloramphenicol 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 Chloramphenicol. |
With both plasmids now in the cell we checked for fluorescence as can be seen in the picture below. | With both plasmids now in the cell we checked for fluorescence as can be seen in the picture below. | ||
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Latest revision as of 21:29, 23 September 2009
University of Aberdeen - Pico Plumber
Results:
BBa_K182001:
Our first successfully constructed BioBrick was K182001, which contains the biobrick BBa_I732820 ( consisting of a RBS, the LacI gene and aTerminator) upstream and the Biobrick BBa_R0040 ( a TetR repressible promoter) downstream.
Since the BioBrick is lacking a promoter upstream of the LacI gene, we were unable to test if it works, We where however able to confirm the sequence.
BBa_K182005
The second BioBrick constructed as part of the LacI latch module was 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 / Chloramphenicol 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 Chloramphenicol.
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.
 Back to LacI-Latch Cloning Strategy
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Continue to Quorum Sensing 
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