Part:BBa_K1962000

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_K1962001). 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.

••••• Parts Collection 2016

This is part of a Part Collection of 17 BioBricks designed by Dundee iGEM 2016. This collection will be useful to teams working with toxins as we have submitted new toxins to the registry. Working with bacterial toxins is difficult due to the risk of toxicity to the chassis, so the corresponding immunity for our toxins were also submitted. We have also submitted these toxins lacking their cytotoxic domains replacing it with a multiple cloning site which will allow for different toxic domains to be fused at the C-terminus and thereby generating a synthetic toxin. In addition, there are three well-characterised promoters that can be used to initiate gene expression at various points in the digestive tract, to enable devices to function within a human or animal. Finally, a lysis cassette was constructed to lyse or burst cells, thus releasing the toxins and destroying the GM bacteria to prevent its release to the environment. This BBa_K1962000 is full-length colicin Ia. It is a control that can be used to compare activity of any synthetic toxin that are generated using this part collection.

Usage and Biology

Colicin Ia 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.

Cloning Strategy

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_asr (BBa_K1231000) and a bile salt sensitive promoter P_acrRA (BBa_K1231001) to generate the composite parts (BBa_K1962015) and (BBa_K1962011), respectively. Next we wanted to clone Colicin Ia downstream of the Ia-Immunity protein however after several attempts we were unable to clone colicin Ia into both of these devices. This may have been due to the fact that this colicin exerted too much of a toxic effect in E. coli and therefore preventing us from obtaining any positive transformants. In order to overcome this problem we decided to clone Colicin Ia downstream of the pBAD promoter, this allowed us to repress expression with 0.5% D-glucose during the cloning steps. However, the only transformants we obtained contained a TAA (stop codon) half way through the gene sequence therefore resulting in a truncated version of Colicin Ia being translated.

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 NgoMIV site found at 132
  Illegal AgeI site found at 1777
  
• 1000
  COMPATIBLE WITH RFC[1000]