Difference between revisions of "Part:BBa K1976052"

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===<h2>Characteristics</h2>===
 
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<h2>Sequence and Features</h2>
 
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Revision as of 14:13, 27 October 2016

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. The protein product from this part was cleaned up and used in different concentrations to test its activity. 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
Im2 immunity protein:
86
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