Part:BBa_K2150022
pTet+RBS+T7RNAP+Ter+pT7+RBS+GFP+DT
In this part, T7 RNA polymerase(T7RNAP) is expressed from pTet which is inducible by tetracycline and its analogs. T7RNAP can activate the T7 promoter, thus producing GFP in large quantity.
Usage and Biology
We design this part to test whether the introduction of T7 polymerase and T7 promoter has the potential to enhance our scavenger's ability to degrade tetracycline in the presence of tetR. In this part, pure GFP is produced, so that the less toxic tetracycline analog anhydrotetracycline (aTc) is not degraded. And then our design can be tested under a constant concentration of Tc (represented by the concentration of aTc). We call this a simulation system.
Characterization
The expression level of GFP rose as the concentration of aTc increased(Fig.1) in the presence of tetR, which indicated that the DNA downstream of T7 promoter was inducible by tetracycline and its analogs.
Comparing to another part (see BBa_K2150014), we found that in the presence of tetR, this part performed better (emitted a higher level of fluorescence) when the concentratiion of aTc was low(Fig.2a, Fig.2b).
However when the concentration of aTc was high, this part inhibited the cell growth(fig.3a), which led to a lower fluorescence intensity than BBa_2150014(Fig.3b). The reason for this is that when pTet was fully induced, T7 RNA polymerase was produced more than enough. Such a large quantity of T7 RNAPs caused a short of resources such as nucleotides in the cell, which inhibited the synthesis of other proteins and eventually led to the inhibition of cell growth. Operating this part on a low or medium copy plasmid may solve this probelm.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3488
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