This construct was created to express Splint R Ligase. 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), SplintR Ligase, and a downstream double terminator (BBa_BB0015).
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<h3> Characterization </h3>
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We used SDS-PAGE to check for SplintR Ligase 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 SplintR Ligase did not express as strongly as we expected, which prompted us to redesign our constructs.
Revision as of 12:57, 25 October 2020
Strong Promoter and RBS SplintR Ligase Expressing Construct
Sequence and Features
Assembly Compatibility:
10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7 Illegal NheI site found at 30
