Part:BBa_K3352007
T7 + RBS Phi29 DNA Polymerase Expressing Construct
Construct Design
This construct was an improved design from our previous construct (BBa_K3352004). This construct consists of a T7 promoter (BBa_J64997), strong RBS, SplintR Ligase, and a downstream double terminator (BBa_BB0015).
PCR Results
Characterization
Seeing that purified Φ29 DNA Polymerase and SplintR Ligase are fundamental to the development of our diagnostic test, we attempted to resolve the issue by introducing a T7 promoter to our construct and expressing our protein with BL21 (DE3) E. coli. DE3 strains contain the chromosomal gene T7 RNA Polymerase which is regulated by a lac promoter. T7 RNA Polymerase has been found to be highly selective and efficient in transcribing only the T7 promoter. Resulting in almost a five-fold faster elongation rate that E. coli RNA Polymerase, T7 would be a much stronger promoter of choice. Thus, by using IPTG during protein synthesis of our BL21 (DE3) E. coli culture, we would effectively produce more T7 RNA Polymerase and significantly increase the production of our enzymes positioned downstream of our T7 promoter.
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 site found at 640
Illegal SapI.rc site found at 1038
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