Part:BBa_K2350020

R0010-B0034-Holin-B0010-B0012-J23106-B0034-Endolysin-B0010-B0012

Endolysin-Holin

　　In this part, we combine endolysin (BBa_K2350018) and holin (BBa_K2350019) for cell lysis. Different from the suicide mechanism used by team PeKing (2014 iGEM Beijing), which belongs to λ lysis system, the types we used are from T4 phage. Besides, the endolysin and holin both are from iGEM released parts with quality assurance.
　　In this composite part, holin functions as an important regulation of the whole suicide mechanism. Therefore, we choose a lactose-induced promoter, BBa_R0010. As for endolysin, we choose a constitutive promoter, BBa_J23106. In our design, we expect our suicide mechanism can reach maximize effect with this perfect combination.

Promoter, RBS, and Terminator

　　For more details of the connected promoter, RBS, and terminator, you can see in BBa_K2350018 for endolysin part and BBa_K2350019 for holin part.

Functional Test

　　The line graph below presents the general trend of O.D.600 value per hour after lactose is added.
　　As the figure shows, the suicide mechanism is induced immediately when lactose is added into the samples. Besides, we can see that the lactose concentration and the OD value of the bacterium with endolysin-holin construct are positively correlated, which means our suicide mechanism does work.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1035
  Illegal NheI site found at 1058
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1362
  Illegal AgeI site found at 1432
  
• 1000
  COMPATIBLE WITH RFC[1000]