Part:BBa_K2938016

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

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]