Part:BBa_K2757002
Bidirectional pTet promoter
Tetracycline inducible promoter system that initiates transcription in both orientations. Ribosome binding sites are present on either side of the promoter.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 86
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterisation
To accurately measure the activity of the promoter, two distinct constructs were used. The negative control construct contains an RFP gene under the control of the bidirectional promoter in one direction and TetR in the other. The expression vector we tested in the assay was identical to the negative control however RFP was replaced with sfGFP, the fluorescence of which was measured at 520nm.
By finding the concentration at which the fluorescence is equal to in the absence of inducer, we can determine that the promoter is insensitive to concentrations below 1 nM.
Our data also was used to determine the leakiness of the promoter. Basal gene expression was obtained by measuring fluorescence in the absence of ATC relative to the negative control. This comparison was critical for isolating that the only contributing factor was the promoter inhibited by TetR.
The steady-state plot for the Bidirectional Promoter was used for data fitting which yielded a value of 1 nM for the maximal transcription rate.
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