Part:BBa_K1223010:Design

pUC-57-P.A.S.E.1

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2179
  Illegal PstI site found at 4832
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2179
  Illegal PstI site found at 4832
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2179
  Illegal BamHI site found at 13
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2179
  Illegal PstI site found at 4832
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2179
  Illegal PstI site found at 4832
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 3495
  Illegal SapI.rc site found at 4577
  

Design Notes

used for amplification and extraction of the P.A.S.E. 1 cassette. the P.A.S.E.1 cassette is cloned into the plasmid backbone using XbaI and BamHI restriction enzymes. we used this part to amplify and extract a large amounts of the P.A.S.E.1 cassette.

the pUC57 is used to overexpress recombinant proteins as well as amplify a gene of interest inside E.coli. the plasmid backbone contains ampicillin resistance gene (b-lactamase) for selection of transformants.

the P.A.S.E.1 cassette 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. pUC57 backbone is a synthetic construct.

holin and lysozyme originates from T4 bacteriophage.

the homology regions were taken from E.coli BL21 genome