Difference between revisions of "Part:BBa K2918023"

(→‎Identification of the DSB protein)
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===Toxicity===
 
===Toxicity===
  
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Our sci-phi29 tool is based on four components of the Φ29 bacteriophage: DNAP, TP, p5 and p6. However, overexpression of these proteins are toxic for the cell. In order to determine the optimal expression levels of the proteins in live cells, we carried out viability assays in E.coli BL21(DE3)PlysS. The results are shown in the graphs below…
  
  

Revision as of 17:03, 3 October 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

Identification of the DSB protein

For identifying 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. A 10-μL reaction consists of 0.5 μL enzym solution, 1 μL ribosome solution, 0.5 μL Green Lyse, 5 nM DNA and RNAse-free milliQ for filling up the volume. The proteins were identified by an 18% SDS-PAGE gel and mass spectrometry.

SDS-PAGE

Figure

An SDS-PAGE was carried out for the DSB protein with 3 different promoter strengths: Wild-Type, 0.5 and 0.1. For a control PURE solution without any DNA was used. The


Mass Spectrometry

In addition to the SDS-PAGE, the identity of the proteins was also confirmed by mass spectrometry. To do this, a sequence unique to the DSB’s amino acid sequence were chosen and screened for their presence in the PURE system. For the p6 the peptide sequences are: GEPVQVVSVEPNTEVYELPVEK and FLEVATVR

Figure

The peak areas of the resulting mass spectrographs shown in Figure ? reflect the occurrence of a given sequence in the sample. The unique peptides that were screened for were only present in each protein expected to contain the sequence.

Hence, it can be concluded that the results were positive and the identity of the proteins could be verified by mass spectrometry.

In Vitro replication

Toxicity

Our sci-phi29 tool is based on four components of the Φ29 bacteriophage: DNAP, TP, p5 and p6. However, overexpression of these proteins are toxic for the cell. In order to determine the optimal expression levels of the proteins in live cells, we carried out viability assays in E.coli BL21(DE3)PlysS. The results are shown in the graphs below…