Composite

Part:BBa_K3790231

Designed by: Chongwen Cao   Group: iGEM21_Fudan   (2021-10-17)


T7 gene product 2 controlled by pBAD/araC


Introduction

2021 Fudan

In order to turn the host bacteria into ‘factories’ that concentrate on manufacturing parts of the virus, some phages have the ability to shut off the endogenous transcription of host bacteria. As for T7 phage, there are three gene products collaboratively realizing the function, which are gene product (gp) 0.7, gp2 and gp5.7[1].

Gp2 is an early phage gene, which means they are transcribed by the host's RNA polymerase at the very beginning of the infection process[2].



Usage and Biology

Gp2 is a small protein that can bind to both the 1.1 domain of σ70 factor and the β’ subunit of the host's RNAP[3], results in effective inhibiting of the transcription initiated by σ70-RNAP complex.

This part can be used to produce gp2 after arabinose induction.

Experimental Results

Gp2 was eliminated by us because gp5.7 is a better choice. For detailed reasons, please visit:

Figure 1. Growth curve of strains with pBAD driven gp2 under different induction level.
Figure 2. GFP/OD600 curve of strains with pBAD driven gp2 under different induction level. The GFP is σ70-driven.

From these two figures, we can conclude that arabinose induction level can't notably influence the effects of pBAD driven gp2 on inhibiting transcription initiated by σ70-RNAP complex and on hindering cell growth.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1205
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1335
    Illegal SapI site found at 961


References

  1. ↑ T7 phage factor required for managing RpoS in Escherichia coli. Tabib-Salazar A,  Liu B,  Barker D,  Burchell L,  Qimron U,  Matthews SJ,  Wigneshweraraj S. Proc Natl Acad Sci U S A, 2018 Jun 5;115(23):E5353-E5362. PMID:29789383
  2. ↑ Roles of the early genes of bacteriophage T7 in shutoff of host macromolecular synthesis. McAllister WT,  Barrett CL. J Virol, 1977 Sep;23(3):543-53. PMID:330878
  3. ↑ Structural and mechanistic basis for the inhibition of Escherichia coli RNA polymerase by T7 Gp2. James E,  Liu M,  Sheppard C,  Mekler V,  Cámara B,  Liu B,  Simpson P,  Cota E,  Severinov K,  Matthews S,  Wigneshweraraj S. Mol Cell, 2012 Sep 14;47(5):755-66. PMID:22819324
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