Difference between revisions of "Part:BBa K1962000"

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Colicin Ia

This sequence codes for the full length Colicin Ia bacteriocin (anti-bacterial toxin). Colicin Ia is a protein that is used by species of E. coli in order to kill closely related species of E. coli in certain circumstances e.g. competition. The producer cell normally contains a protein which confers immunity to this toxin, which in this case is (BBA_). And this protein is believed to form a complex with the cytotoxic domain of the bacterocin rendering it inactive. When this bacteriocin is secreted the immunity protein dissociates from the complex leaving the cytotoxic domain active resulting in the pore forming activity of this bacteriocin returning and having effect on the target cells. The pore forming activity of this bacteriocin results in the depolarisation of the bacterial inner membrane resulting in the collapse of the protonmotive force.

Usage and Biology

Colicin iA is a protein that is used by species of E. coli in order to kill closely related species of E. coli in certain circumstances e.g. competition. This protein is synthesised on the ribosome and translocation occurs by the TonB system as this is a group B colicin. Expression of these proteins is tightly regulated by the REcA and LexA proteins involved in the SOS response. The expression of Colicins can be inhibited by the binding of LexA to the SOS promoter and this action is reversed by the binding of RecA to LexA. This colicin uses two Cir receptors in order to enter the cell. One for binding and another for translocation. The TonB protein forms a complex with the other cell membrane proteins, Exb8 and ExbD and this complex provides the energy required for the translocation of the cytotoxic domain into the cell.

Below is the structure of Colicin iA, in green is the receptor binding domain, in red the translocation domain and in blue the cytotoxic domain. This was cloned into pSB1C3 with the Biobrick prefix and suffix. We attempted to clone Colicin Ia downstream of the composite part (BBa) in order to control expression of Colicin Ia in response to different pH conditions. However, after many attempts we were unable to clone this downstream of the Pasr-Ia-Im composite brick. We then decided to clone Colicin Ia downstream of a more controllable promoter, so we again attempted to clone Colicin Ia downstream of the pBAD promoter. During the cloning steps we repressed the promoter by the addition of 0.5% D-Glucose. However the only transformants we obtained contained a stop codon.

Sequence and Features