Difference between revisions of "Part:BBa K2938015"
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PR1 - RBS - Cry11Aa(Strep tag) - RBS - deGFP - T500 Terminator
 
PR1 - RBS - Cry11Aa(Strep tag) - RBS - deGFP - T500 Terminator
  
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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.
+
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.
  
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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.
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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===
 
===Characterization===
  
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Western of Cry11Aa subunit with anti-Strep antibody. proving the translation of the toxin subunit on the plasmid.
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Western of Cry11Aa subunit with anti-Strep antibody. Proving the translation of the toxic subunit in the plasmid.
  
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and confocal microscope photo showing our transgenic bacteria presence in the mosquitoes larva
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A Confocal microscope photo showing our transgenic bacteria present in a mosquito larva.
  
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[[File:Larva GFP.png|200px|thumb|left|Mosquitoes' larva that was fed with serratia marcescens 274 expressing plasmid with Cry11Aa and deGFP. confocal microscope (x10)]]
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[[File:Larva GFP.png|200px|thumb|left|Mosquito larva that was fed with Serratia marcescens 274 expressing the plasmid with Cry11Aa and deGFP. Confocal microscope (x10)]]
 
[[File:Cry11Aa_Western.png|200px|thumb|right|Western Blot of Bacteria expressing Cry11Aa and deGFP – proof of Cry11Aa expression]]
 
[[File:Cry11Aa_Western.png|200px|thumb|right|Western Blot of Bacteria expressing Cry11Aa and deGFP – proof of Cry11Aa expression]]
  
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===Experiance===
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===Experience===
 
In our plasmids, we placed the deGFP closer to the terminator.
 
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)
+
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).  
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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).  
  
 
[[File:Fluorescence Plasmids Serratia.png|200px|thumb|left|Fluorescence levels of Serratia expressing our different plasmids]]
 
[[File:Fluorescence Plasmids Serratia.png|200px|thumb|left|Fluorescence levels of Serratia expressing our different plasmids]]
  
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We made toxicity assays, In order to check how effective and toxic are our plasmids.
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We carried out toxicity assays in order to check if our plasmids are effective and toxic to the mosquito larvae.
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We hatched mosquitoes untreated eggs in water containing Serratia bacteria expressing the different plasmids.
+
We hatched untreated mosquito eggs in water containing our Serratia bacteria expressing the different plasmids.
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Then we counted the live larva 48 hours after treatment.
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Then we counted the live larvae 24 and 48 hours after treatment.
  
  

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


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]