Device

Part:BBa_K2938016

Designed by: Assaf Vital   Group: iGEM19_BGU_Israel   (2019-10-09)
Revision as of 13:24, 12 October 2019 by Assafi (Talk | contribs) (→‎Experiance)

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Cry11Aa (Strep tag) + P20 (His tag) + deGFP Device

PR1 - RBS - Cry11Aa (Strep tag) - RBS - P20 (His tag) - RBS - deGFP - T500 Terminator

This plasmid was planned on a pBEST plasmid backbone. It contains larvicidal activity, as it containing toxic subunit of the bacillus thuringiensis israelensis (BTI) pBtoxis plasmid which is toxic to mosquitoes larva.

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 and P20 subunits with anti-Strep and anti-His antibodies. proving the translation of the toxin subunit and chaperone on the plasmid.

Western Blot of Bacteria expressing Cry11Aa and deGFP – proof of Cry11Aa expression
Western Blot of Bacteria expressing Cry11Aa, P20 and deGFP – proof of P20 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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1870
    Illegal EcoRI site found at 2426
    Illegal XbaI site found at 2116
    Illegal XbaI site found at 2761
    Illegal SpeI site found at 982
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1870
    Illegal EcoRI site found at 2426
    Illegal NheI site found at 56
    Illegal NheI site found at 105
    Illegal SpeI site found at 982
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1870
    Illegal EcoRI site found at 2426
    Illegal XhoI site found at 3494
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1870
    Illegal EcoRI site found at 2426
    Illegal XbaI site found at 2116
    Illegal XbaI site found at 2761
    Illegal SpeI site found at 982
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1870
    Illegal EcoRI site found at 2426
    Illegal XbaI site found at 2116
    Illegal XbaI site found at 2761
    Illegal SpeI site found at 982
  • 1000
    COMPATIBLE WITH RFC[1000]


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