Difference between revisions of "Part:BBa K3352006"
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<partinfo>BBa_K3352006 short</partinfo> | <partinfo>BBa_K3352006 short</partinfo> | ||
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| + | <h3> Construct Design </h3> | ||
This construct was an improved design from our previous construct (BBa_K3352004). This construct consists of a T7 promoter (BBa_I712074), strong RBS, SplintR Ligase, and a downstream double terminator (BBa_BB0015). | This construct was an improved design from our previous construct (BBa_K3352004). This construct consists of a T7 promoter (BBa_I712074), strong RBS, SplintR Ligase, and a downstream double terminator (BBa_BB0015). | ||
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| + | <h3> Characterization </h3> | ||
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. | 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. | ||
Revision as of 15:53, 18 October 2020
T7 + RBS SplintR Ligase Expressing Construct
Construct Design
This construct was an improved design from our previous construct (BBa_K3352004). This construct consists of a T7 promoter (BBa_I712074), strong RBS, SplintR Ligase, and a downstream double terminator (BBa_BB0015).
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]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 835
- 1000COMPATIBLE WITH RFC[1000]