Difference between revisions of "Part:BBa K2938015"

Latest revision as of 13:23, 12 October 2019

Cry11Aa (Strep tag) + deGFP Device

PR1 - RBS - Cry11Aa(Strep tag) - RBS - deGFP - T500 Terminator

This plasmid was planned on a pBEST plasmid backbone. It contains larvicidal activity, as it contains a toxic subunit of the Bacillus thuringiensis israelensis (Bti), a pBtoxis plasmid which is toxic to mosquito larvae.

The plasmid was constructed using Gibson Assembly, then was incorporated into E. coli BL21 and DH5a using Heat Shock, and into Serratia marcescens 274 using Electroporation.

Characterization

Western of Cry11Aa subunit with anti-Strep antibody. Proving the translation of the toxic subunit in the plasmid.

A Confocal microscope photo showing our transgenic bacteria present in a mosquito larva.

Mosquito larva that was fed with Serratia marcescens 274 expressing the plasmid with Cry11Aa and deGFP. Confocal microscope (x10)

Western Blot of Bacteria expressing Cry11Aa and deGFP – proof of Cry11Aa expression

Experience

In our plasmids, we placed the deGFP closer to the terminator. The presence of the GFP signal proves the expression of the previous subunits on the plasmid and it allows the detection of the transgenic bacteria easily (in mosquitoes).

Here we can see the level of fluorescence of liquid starters from Serratia expressing the different types of plasmid we created. (BBa_K2938014, BBa_K2938015, BBa_K2938016).

Fluorescence levels of Serratia expressing our different plasmids

We carried out toxicity assays in order to check if our plasmids are effective and toxic to the mosquito larvae. We hatched untreated mosquito eggs in water containing our Serratia bacteria expressing the different plasmids. Then we counted the live larvae 24 and 48 hours after treatment.

Toxicity assay results, Our plasmids show larvicidal activity