Difference between revisions of "Part:BBa K2918017"
(→‎Toxicity)
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   <li style="display: inline-block;"> [[File:T--TUDelft--0.1tp-nomM.png|thumb|none|444px|<b>Figure 3A:</b> The growth curve of TP under a 0.1 T7 promoter in hours with 1 mM IPTG induction]] </li>
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   <li style="display: inline-block;"> [[File:T--TUDelft--TPnoiptg.png|thumb|none|444px|<b>Figure 3A:</b> The growth curve of phi29 TP under different promoter strengths (weak, medium, Wild-Type) with no IPTG induction]] </li>
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   <li style="display: inline-block;"> [[File:T--TUDelft--0.1tp-1mM.png|thumb|none|444px|<b>Figure 3B:</b> The growth curve of TP under a 0.1 T7 promoter in hours with 1 mM IPTG induction]] </li>
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     <li> [[File:T--TUDelft--0.1tp-10mM.png|thumb|center|444px|<b>Figure 3C:</b> The growth curve of TP under a 0.1 T7 promoter in hours with 10 mM IPTG inductionn]] </li>
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   <li style="display: inline-block;"> [[File:T--TUDelft--tp1iptg.png|thumb|none|444px|<b>Figure 3B:</b> The growth curve of phi29 p6 under different promoter strengths (weak, medium, Wild-Type) with 1 mM IPTG induction]] </li>
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     <li> [[File:T--TUDelft--tp10iptg.png|thumb|center|444px|<b>Figure 3C:</b>The growth curve of phi29 p6 under different promoter strengths (weak, medium, Wild-Type) with 10 mM IPTG induction]] </li>
 
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Revision as of 21:22, 6 December 2019


Weak T7 promoter - Universal RBS - Φ29 TP - WT T7 terminator

This part consists of a 0.1 T7 promotor, a universal Ribosome Binding Site (RBS), a Coding DNA Sequence (CDS) coding for the TP and a Wild Type T7 terminator.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 227
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 547
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

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

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: The growth curve of phi29 TP under different promoter strengths (weak, medium, Wild-Type) with no IPTG induction
  • Figure 3B: The growth curve of phi29 p6 under different promoter strengths (weak, medium, Wild-Type) with 1 mM IPTG induction
  • Figure 3C:The growth curve of phi29 p6 under different promoter strengths (weak, medium, Wild-Type) with 10 mM IPTG induction