Part:BBa_K2918023

Weak T7 promoter - Universal RBS - Φ29 DSB (p6) - WT T7 terminator

This part consists of a T7 promotor, a universal Ribosome Binding Site (RBS), a Coding DNA Sequence (CDS) coding for the DSB p6 and a Wild Type T7 terminator.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 292
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 68
  Illegal BsaI.rc site found at 94
  

Overview

This protein is one of the 4 components needed for orthogonal replication

Characterization

For characterizing our constructs we used PURE(Protein synthesis Using Recombinant Elements) system. This is an E.coli based cell-free protein synthesis system and it contains all the elements to make in vitro translation-transcription possible. Quantification of the DSB protein was done with green Lyse and then run on a 18% SDS-PAGE gel (see figure).

Protein gel

Mass Spectrometry The same mixture of plasmid, green lyse and PURE system was also run on a Mass Spectrometer, this only indicates that the unique peptide fragments of this protein are there.

Plasmid is also checked by sequencing (where there mutations)