Part:BBa_K624044

Safety Flag

The iGEM Safety and Security Committee has placed a Red Flag on this part. This part presents safety risks beyond what is normal for the Registry. Researchers who plan to acquire and use this part should take special care to ensure they use it safely and responsibly. Contact safety [AT] igem [DOT] org with any questions.

Reason: Listeriolysin and Invasin parts

If you are an iGEM team, you must submit a Check-In before acquiring and using this part! See the 2021 Safety Page for more information.

pYMB essentials + RBS + LLO + RBS + Invasin + RBS + ECFP

ori + Pmsp1 + rep + RBS + LLO + RBS + Invasin + ECFP

pYMB (BBa_K624004) is described to contain the ori (origin of replication), rep gene (required for replication) of pMGT. Once the synthetic work had been done, pYMB is constructed by equipping the ori, the appropriate promoter for AMB-1 (Pmms16 and Pmsp3 as our candidate) and rep gene on the commercial plasmid pUG19 which was revised as the expression of Biobrick backbone pSB1A1 with promoter Pmsp1. The constructed vector is capable of replicating within both E. coli and AMB-1, fully sufficing a competent shuttle vector for genetic engineering the magnetotactic bateria.

Listeriolysin O (LLO) is a hemolysin produced by the bacterium Listeria monocytogenes, a pathogen that is responsible for listeriosis. LLO is activated within phagosomes of cells that have phagocytosed L. monocytogenes cells, and lyses the membrane of phagosome. The bacteria is then able to escape into the cytosol without damaging the plasma membrane, and grow intracellularly. This would result in the protection from extracellular immune system.

Invasin is an outer membrane protein that allows bacteria to penetrate mammalian cells. It is from Yersinia enterocolitica. Invasin binds to multiple Beta 1 chain integrin receptors on mammalian cells with high affinity, which induces endocytosis and internalization.

All of the ribosome binding sites are from Pmsp3, the strongest promoter of AMB-1.(BBa_K624012)

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 2567
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3573
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 898
  Illegal NgoMIV site found at 4190
  
• 1000
  COMPATIBLE WITH RFC[1000]