Part:BBa_K1976052

Colicin E2 DNase domain and immunity protein Im2 generator with anderson promoters

Generator of colicin E2 DNase domain BBa_K1976048 and immunity protein Im2 BBa_K1976026.

Figure 1: The structure of the Im2 immunity protein and Colicin E2 in a complex.

Usage

This part can be used to produce colicin without killing the host cell, as the immunity protein is produced more than equimolar to the DNase fragment. If BBa_K1976028 is used instead of BBa_K1976027, it can function as a amber-suppression inducible killswitch.

Characteristics

Figure 2: SDS-PAGE of the Colicin E2 DNase domain, the blue arrow marks the band of the expression. From left to right: Top10 E. coli, Top10 E. coli transformed with BBa_K1976052 and Top10 E. coli transformed with BBa_K1976053.

Figure 3: 1% agarose gel electrophoresis, stained with HD-green.

From left to right: pSB1C3 alone, pSB1C3 in the lysate buffer 1x, pSB1C3 in the lysate buffer 3x, pSB1C3 incubated with lysate of Top10 transformed with BBa_K1976052 1x, pSB1C3 incubated with lysate of Top10 transformed with BBa_K1976052 3x, pSB1C3 incubated with lysate of Top10 transformed with BBa_K1976053 1x, pSB1C3 incubated with lysate of Top10 transformed with BBa_K1976053 3x.

Molecular Weight	Colicin E2 Dnase domain 15.2 kDa Immunity Protein 10.0 kDa
Residues	Colicin E2 Dnase domain 133
pI	Colicin E2 Dnase domain 9.06 Immunity Protein 4.66
[Data taken created in Snapgene and http://isoelectric.ovh.org/calculate.php]

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal NheI site found at 477
  Illegal NheI site found at 500
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 681
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 89
  
• 1000
  COMPATIBLE WITH RFC[1000]

[1] Cascales et al, Colicin Biology, Microbiology and Molecular Biology Reviews, vol. 71, pp. 158-229, 2007