Part:BBa_K1223001

P.A.S.E. 1 cassette

A DNA cassette that undergo homologous recombination into E.coli genome that contains homologous regions at each end, Kanamicin resistance gene (KanR) and death operon containing Lambda holin and lysozime genes and controlled by a Lambda cI regulated promoter

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 13
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

This part was designed to fulfill the self destruct system of P.A.S.E 1. It contains a toxin system based on phage lysis system of holin (BBa_K112805) and lysozyme (BBa_K112301). Holin protein causes "pores" in the inner membrane, which allows lysozyme to access and break down the peptidoglycan of the cell wall, causing lysis and eventually death. The toxins are regulated by cI regulated promoter (BBa_R0051). This part is designed to integrate into the cell's genome via homologous recombination and therefore it contains homologous regions at its ends. Kanamycin resistance was added for selectivity. Therefore, when transforming in bacteria only the cells that have gone through double recombination with the insert will survive.

The part was characterized through sequencing and restriction digest with BamHI and EcorRV.

lane1: undigested pUC57-P.A.S.E.1 cassette

lane2: pUC57-P.A.S.E.1 cassette digested with BamHI and EcorRV.

lane3: DS5000 Ladder

Source

part is synthetic.

holin and lysozyme originates from T4 bacteriophage.

the homology regions were taken from E.coli BL21 genome