Difference between revisions of "Part:BBa K2918023"
(→‎Characterization)
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===Characterization===
 
===Characterization===
  
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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.  
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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).  
  
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Protein gel + Mass Spectrometry + transformation + sequencing(gotag check pcr)
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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.
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 +
 
 +
 
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Plasmid is also checked by sequencing (where there mutations)
  
 
===Toxicity===
 
===Toxicity===

Revision as of 19:40, 28 September 2019


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)

Toxicity