Device
Cry11Aa Pl

Part:BBa_K2938015

Designed by: Assaf Vital   Group: iGEM19_BGU_Israel   (2019-10-09)
Revision as of 13:14, 12 October 2019 by Assafi (Talk | contribs) (→‎Characterization)


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













Experiance

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 type of plasmid we created. (BBa_K2938014, BBa_K2938015, BBa_K2938016).

Fluorescence levels of Serratia expressing our different plasmids

We made toxicity assays, In order to check how effective and toxic are our plasmids. We hatched mosquitoes untreated eggs in water containing Serratia bacteria expressing the different plasmids. Then we counted the live larva 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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