Difference between revisions of "Part:BBa K2938018"
 
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[[File:ToxinTable.png]]
 
[[File:ToxinTable.png]]
  
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In order to build this plasmid, we constructed three initial plasmids [BBa_K2938014, BBa_K2938015, BBa_K2938016] which can build this final plasmid using gibson assembly.
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In order to build this plasmid, we constructed three initial "minimal" plasmids [BBa_K2938014, BBa_K2938015, BBa_K2938016] which would assist in building this final plasmid using Gibson Assembly. Our 3 initial "minimal" plasmids showed toxicity against the mosquito larvae (which you can see under "Experience" page of the plasmids), and the final plasmid should show greater toxicity due to synergistic activity of the different subunits.
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Our 3 initial plasmids showed toxicity against the mosquito larva (which you can see under experiences page of the plasmids), and the final plasmid should show better toxicity due to synergistic activity of the different subunits.
+
  
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During the gibson assembly of the initial plasmids we added tags to each of the subunit in order to see it's expression using western blot, and we placed deGFP downstream of the polycistronic plasmids, closest to the terminator in order to follow our GFP marker in confocal microscope meaning the upstream toxic genes were also expressed.
+
During the Gibson Assembly of the initial "minimal" plasmids, we added tags to each of the subunits in order to see their expression using Western Blot. We also placed deGFP downstream of the polycistronic plasmid, near the terminator in order to follow our GFP marker using a Confocal microscope, which would help determine if the upstream toxic genes were also expressed.
  
 
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Latest revision as of 13:24, 12 October 2019


Polycistronic BTI Toxin

PR1 - RBS - Cry4Ba (HA tag) - RBS - Cry11Aa (Strep tag) - RBS - Cyt1Aa (Strep tag) - RBS - P20 (His tag) - RBS - deGFP - T500 Terminator.

We planned this plasmid to be the most toxic to the Aedes aegypti mosquito larvae. Containing the most toxic Bacillus thuringiensis subsp. israelensis (Bti) subunits. ToxinTable.png

In order to build this plasmid, we constructed three initial "minimal" plasmids [BBa_K2938014, BBa_K2938015, BBa_K2938016] which would assist in building this final plasmid using Gibson Assembly. Our 3 initial "minimal" plasmids showed toxicity against the mosquito larvae (which you can see under "Experience" page of the plasmids), and the final plasmid should show greater toxicity due to synergistic activity of the different subunits.

During the Gibson Assembly of the initial "minimal" plasmids, we added tags to each of the subunits in order to see their expression using Western Blot. We also placed deGFP downstream of the polycistronic plasmid, near the terminator in order to follow our GFP marker using a Confocal microscope, which would help determine if the upstream toxic genes were also expressed.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 1846
    Illegal EcoRI site found at 2142
    Illegal EcoRI site found at 6316
    Illegal EcoRI site found at 6627
    Illegal XbaI site found at 3820
    Illegal XbaI site found at 5510
    Illegal SpeI site found at 958
    Illegal SpeI site found at 4509
    Illegal PstI site found at 2324
    Illegal PstI site found at 5976
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1846
    Illegal EcoRI site found at 2142
    Illegal EcoRI site found at 6316
    Illegal EcoRI site found at 6627
    Illegal NheI site found at 56
    Illegal NheI site found at 105
    Illegal NheI site found at 2093
    Illegal NheI site found at 2208
    Illegal SpeI site found at 958
    Illegal SpeI site found at 4509
    Illegal PstI site found at 2324
    Illegal PstI site found at 5976
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1846
    Illegal EcoRI site found at 2142
    Illegal EcoRI site found at 6316
    Illegal EcoRI site found at 6627
    Illegal BglII site found at 6274
    Illegal BamHI site found at 5653
    Illegal XhoI site found at 7655
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 1846
    Illegal EcoRI site found at 2142
    Illegal EcoRI site found at 6316
    Illegal EcoRI site found at 6627
    Illegal XbaI site found at 3820
    Illegal XbaI site found at 5510
    Illegal SpeI site found at 958
    Illegal SpeI site found at 4509
    Illegal PstI site found at 2324
    Illegal PstI site found at 5976
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 1846
    Illegal EcoRI site found at 2142
    Illegal EcoRI site found at 6316
    Illegal EcoRI site found at 6627
    Illegal XbaI site found at 3820
    Illegal XbaI site found at 5510
    Illegal SpeI site found at 958
    Illegal SpeI site found at 4509
    Illegal PstI site found at 2324
    Illegal PstI site found at 5976
    Illegal NgoMIV site found at 4632
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2614