Composite

Part:BBa_K3352004

Designed by: Hannah Hsu   Group: iGEM20_TAS_Taipei   (2020-10-18)
Revision as of 14:35, 18 October 2020 by Registry (Talk | contribs)

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Strong Promoter and RBS SplintR Ligase Expressing Construct

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).

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.


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
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 851
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None