Difference between revisions of "Part:BBa K3352005"
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<partinfo>BBa_K3352005 short</partinfo> | <partinfo>BBa_K3352005 short</partinfo> | ||
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| + | <h3> Construct Design </h3> | ||
This construct was created to express Φ29 DNA Polymerase. Sequences used for the promoter, RBS, and terminator came from parts included in the iGEM Registry. This construct consists of a strong promoter and a strong RBS combination (BBKa_K880005), Φ29 DNA Polymerase, and a downstream double terminator (BBa_BB0015). | This construct was created to express Φ29 DNA Polymerase. Sequences used for the promoter, RBS, and terminator came from parts included in the iGEM Registry. This construct consists of a strong promoter and a strong RBS combination (BBKa_K880005), Φ29 DNA Polymerase, and a downstream double terminator (BBa_BB0015). | ||
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| + | <h3> Characterization </h3> | ||
We used SDS-PAGE to check for Φ29 DNA Polymerase expression in E. coli. We prepared overnight DH5⍺ E. coli cultures and obtained OD600 readings to dilute cultures to standardized populations. We grew the cultures to log phase, and lysed cells with xTractor Lysis Buffer (Takara Bio). We purified our His-tagged proteins using Ni sepharose affinity chromatography, then ran our prepared samples through a nickel column in order to purify out our his-tagged proteins. However, the SDS-PAGE results showed that our Φ29 DNA Polymerase did not express as strongly as we expected, which prompted us to redesign our constructs. | We used SDS-PAGE to check for Φ29 DNA Polymerase expression in E. coli. We prepared overnight DH5⍺ E. coli cultures and obtained OD600 readings to dilute cultures to standardized populations. We grew the cultures to log phase, and lysed cells with xTractor Lysis Buffer (Takara Bio). We purified our His-tagged proteins using Ni sepharose affinity chromatography, then ran our prepared samples through a nickel column in order to purify out our his-tagged proteins. However, the SDS-PAGE results showed that our Φ29 DNA Polymerase did not express as strongly as we expected, which prompted us to redesign our constructs. | ||
Revision as of 15:52, 18 October 2020
Strong Promoter and RBS phi29 DNA Polymerase Ligase Expressing Construct
Construct Design
This construct was created to express Φ29 DNA Polymerase. Sequences used for the promoter, RBS, and terminator came from parts included in the iGEM Registry. This construct consists of a strong promoter and a strong RBS combination (BBKa_K880005), Φ29 DNA Polymerase, and a downstream double terminator (BBa_BB0015).
Characterization
We used SDS-PAGE to check for Φ29 DNA Polymerase expression in E. coli. We prepared overnight DH5⍺ E. coli cultures and obtained OD600 readings to dilute cultures to standardized populations. We grew the cultures to log phase, and lysed cells with xTractor Lysis Buffer (Takara Bio). We purified our His-tagged proteins using Ni sepharose affinity chromatography, then ran our prepared samples through a nickel column in order to purify out our his-tagged proteins. However, the SDS-PAGE results showed that our Φ29 DNA Polymerase did not express as strongly as we expected, which prompted us to redesign our constructs.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 656
Illegal SapI.rc site found at 1054