Part:BBa_K3352005

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

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 656
  Illegal SapI.rc site found at 1054