Translational_Unit

Part:BBa_K1593667

Designed by: Juntao Yu   Group: iGEM15_USTC   (2015-08-14)
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Reason: envelope protein

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T7-SCVE

This circuit containing strong expression of SCVE regulated by T7, which will significantly improve bacterial permeability.

Description

T7 promoter is very specific promoter which is transcribed only by specific T7 RNA polymerase. Usually this promoter is used in expression systems where T7 promoter is cotransfected with T7 RNA polymerase. That ensures strong transcription of desired genes.
SCVE, the abbreviation of SARS Caronavirus Envolope protein,is a kind of effective viroporin(viroporins are similar to porins in that they oligomerize within the membrane to form pores and this oligomerization often occurs between hydrophobic, transmembrane and alpha-helical domains of the proteins) within 76 residues that is found normally pentamerized in bacteria. SCVE is permeable for many small molecules, which is originately designed for apoptosis of cells triggered by virus. Implementation of SCVE in ROSE may potentially improve the permatilibty of small molecules including antibiotics.
We connected these two parts together into T7-SCVE to enhance the expression of SCVE.

Experimental Result

Construction of plasimid with T7-SCVE (BBa_K1593667) . We finally ligate the T7 with SCVE and got T7-SCVE (BBa_K1593667).

The result of the polymerase chain reaction (PCR) of T7-SCVE

Growth Characterization

We firstly characterized the growth rate of genetically modified CACCI along with wild type BL21 using optical density at 600 nm, which is called OD 600, in order to demonstrate that the overexpression of porin(OprF with T7,BBa_K1593209) or viroporin(SCVE with T7, (BBa_K1593667) won't severely affect the growth of bacteria, at least it won't significantly inhibit bacteria growth and become as kill switch.

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ONPG Assay

This is to show the absorption capability of the bacteria is improved.

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Reference

1. T. Mattila-Sandholm et al. Fluorometric assessment of Gram-negative bacterial permeabilization. Journal of Applied Microbiology 2000, 88, 213–219
2. Scott Banta et al. Genetic Manipulation of Outer Membrane Permeability: Generating Porous Heterogeneous Catalyst Analogs in Escherichia coli dx.doi.org/10.1021/sb400202s ACS Synth. Biol. 2014, 3, 848−854 Sequence and Features BBa_K1593667 SequenceAndFeatures

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