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 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 rises as the concentration of aTc increases(Fig.1), in the presence of tetR, which indicates the DNA downstream of T7 promoter is inducible by tetracycline and its analogs.
Comparing to another part (see BBa_K2150014), in the presence of tetR, this part performs better (emits a higher level of fluorescence) when the concentratiion of atc is low(Fig.2a, Fig.2b).
However when the concentration of aTc is high, this part will inhibit the cell growth(fig.3a), which leads to a weaker fluorescence intensity than BBa_2150014(Fig.3b). The reason for this is that when pTet is fully induced, T7 RNA polymerase is 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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