Difference between revisions of "Part:BBa K1962001"

Revision as of 21:50, 22 October 2016

Immunity Protein Im-Ia

This biobrick codes for the Immunity Protein for Colicin Ia (BBa_K1962000). Colicin Ia is a channel-forming bacteriocin that depolarizes the cytoplasmic inner membrane of target bacteria, leading to dissipation of cellular energy. This Immunity Protein is tightly linked to its specific Colicin bacteriocin domain to protect the colicinogenic cell from the cytotoxic activity of the colicin.

Usage and Biology

The cell-killing potential of a pore-forming colicin is remarkable in that one molecule kills one cell. Equally remarkable is the protection system that operates at 104 to 107 times the concentration of colicin that would kill a nonimmune cell. This protective mechanism is achieved by a small polypeptide of 11 to 18 kDa, called the immunity protein, encoded by the same plasmid as colicin. The immunity proteins confer upon cells protection against the colicin they produce but not against heterologous colicins with identical modes of action, even those with considerable sequence similarity. Interestingly, immunity proteins are required to protect the cell against the action of exogenous colicin, probably produced by its neighbors, but are not required to protect it from internal colicin, since the polarity of the transmembrane potential is opposite to that required to open the pore. The specificity of colicins with respect to immunity is determined by the C-terminal pore-forming domains.

Cloning Strategy

Our idea was to generate a device that would express Colicin Ia in response to low pH and bile salts so in order to protect the host cells from the toxic activity of Colicin iA we began by cloning the iA-Immunirty protein downstream of a pH sensitive promoter P_asr (BBa_K1231000) and a bile salt sensitive promoter P_acrRA (BBa_K1231001) to generate the composite parts (BBa_K1962015) and (BBa_K1962011), respectively. However, after successfully cloning the iA-Immunity protein we were unable to clone the Colicin iA downstream of the promoter and the immunity.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 46
Illegal AgeI site found at 253
1000
COMPATIBLE WITH RFC[1000]

@@ Line 11: / Line 11: @@
 <u>Cloning Strategy</u>
-Our aim was to create a device which would express Colicin Ia in response to pH and bile salts. We cloned the Ia-Immunity protein downstream of a pH sensitive promoter P<sub>asr</sub> (<partinfo>BBa_K1231000</partinfo>) and a bile salt sensitive promoter P<sub>acrRA</sub> (<partinfo>BBa_K1231001</partinfo>) to generate the composite parts (<partinfo>BBa_K1962015</partinfo>) and (<partinfo>BBa_K1962011</partinfo>), respectively.
+Our idea was to generate a device that would express Colicin Ia in response to low pH and bile salts so in order to protect the host cells from the toxic activity of Colicin iA we began by cloning the iA-Immunirty protein downstream of a pH sensitive promoter P<sub>asr</sub> (<partinfo>BBa_K1231000</partinfo>) and a bile salt sensitive promoter P<sub>acrRA</sub> (<partinfo>BBa_K1231001</partinfo>) to generate the composite parts (<partinfo>BBa_K1962015</partinfo>) and (<partinfo>BBa_K1962011</partinfo>), respectively. However, after successfully cloning the iA-Immunity protein we were unable to clone the Colicin iA downstream of the promoter and the immunity.
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