Composite
WT DNAP

Part:BBa_K2918022

Designed by: TUDelft2019   Group: iGEM19_TUDelft   (2019-09-27)
Revision as of 22:15, 6 December 2019 by Weronika77 (Talk | contribs) (→‎Toxicity)


WT T7 promoter - Universal RBS - Φ29 DNAP - 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 1770
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 60
    Illegal BsaI.rc site found at 86

The construct is confirmed by sequencing and there are no mutations.

Overview

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

Characterization

Protein gel + Mass Spectrometry + transformation + sequencing(gotag check pcr)

Toxicity

Our Sci-Phi 29 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.

  • Figure 3A: Normalized maximum growth rate of phi29 DNAP under different promoter strengths (weak, medium, Wild-Type) with no IPTG induction
  • Figure 3B: Normalized maximum growth rate of phi29 DNAP under different promoter strengths (weak, medium, Wild-Type) with 1 mM IPTG induction
  • Figure 3C: Normalized maximum growth rate of phi29 DNAP under different promoter strengths (weak, medium, Wild-Type) with 10 mM IPTG induction


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Categories
Parameters
None