Coding
gp2

Part:BBa_K3790085

Designed by: Chongwen Cao   Group: iGEM21_Fudan   (2021-10-01)
Revision as of 23:00, 21 October 2021 by Chongwen (Talk | contribs)


T7 phage gene product 2, an E.coli transcription inhibitor


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.

Experimental Results

Gp2 was eliminated by us because gp5.7 is a better choice. For more details, please visit: https://parts.igem.org/wiki/index.php?title=Part:BBa_K3790050

Figure 1. Oligo assembly by PCR. It is generally used to construct completely new or special-purpose DNA. This method may have the disadvantage of a high mutation rate when operated. Once, we had to sequence nine clones of the same construct to get a single correct one. The reason for this is most likely due to complex annealing and amplification. We suggest to have 10-15 rounds amplification without F1 or R1 primer, then add those two primers to have another 25 rounds. Must use high-fidelity enzymes for this method. Due to the pricing, we always use 60bp primers, 58 overlapping annealing temperature, to assemble 300-500bp DNA fragment.

The length of albA1 DNA was 288 bp, which is approximately 300 bp after adding homology arms to both ends for PCR cloning. We isolated the DNA of interest by gel extraction for subsequent reactions.

Figure 2. Assembled DNA binding proteins, albA1, S.ssb, E.ssb. The first lane was loaded with DNA ladder, sizes were marked on the image. The brightest band of 750 bp was about 100 ng, and other bands about 50 ng. Lanes with correct sized amplified DNA were labeled. After PCR cloning, several bacterial clones were picked, grew into cultures and sent for Sanger sequencing. Then, we verified the sequencing results, and used the correct ones for further experiments.


Reference

  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


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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//cds/transcriptionalregulator
Parameters
None