Part:BBa_K315048:Experience

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Applications of BBa_K315048

Reporter Placement: Results from past Davidson and Missouri Western research hinted at the presence of a transcriptional terminator within the TetA gene. Any gene located downstream of the 3’ end of the TetA gene did not appear to be expressed. This gene codes for an effluent pump that physically removes the antibody tetracycline from the cell. To test for the presence of this terminator, we created a construct with a fluorescent protein gene downstream of TetA. If TetA really does block transcription, we would expect to see no fluorescence despite induction of the promoter that precedes the construct (pLac+TetA+RFP).

We conducted experiments with two constructs (pLac+TetA+RFP (K315003) and pLac+RFP+TetA (K315048)) to determine the influence of gene order on fluorescence. In our experiments, pLac+RFP+TetA showed significantly higher fluorescence than pLac+TetA+RFP.

The fluorescence of both constructs was normalized with respect to pLac+RBS+RFP. Even with induced with IPTG (0.5 mM), pLac+TetA+RFP showed no significant increase in fluorescence. This data continues to support the presence of a transcription terminator in TetA. In summary, we recommend placing reporters before the TetA in any constructs being engineered.

Influence of media on fluoroscence:

In further tests, we observed a significant decrease in fluorescence when the cell type pLac+RFP+TetA was grown in tetracycline (50ug/mL) media. The cells continued to live in the tetracycline media with a marginal drop in cell density. However, the cells displaced considerably less fluorescence in this media even when induced with IPTG. This unexpected result suggests that tetracycline media hinders cell fluorescence.

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