Generator
ColE2

Part:BBa_K131000

Designed by: Kevin McLeod   Group: iGEM08_Calgary_Wetware   (2008-07-22)

ColE2 Operon

This part contains the ColE2 operon in its entirety.

Usage and Biology

A colicin is an antibiotic protein produced by some E. coli strains that is active against other E. coli not possessing the plasmid (which contains an immunity gene). Colicin E2 is a DNAse, which enters the cell through the BtuB receptor.

This operon is under the control of the SOS promoter from E. coli and is repressed under normal conditions. This promoter is activated upon DNA damage by the SOS response, which can be initiated in the lab by the addition of some dilute antibiotics, such as mitomycin C. When active, the promoter allows expression of both the colicin itself (K131004) and the immunity gene (K131003). After the immunity gene there is a terminator, followed directly by the lysis gene (K131005). Because of this, the lysis gene is only expressed when RNA polymerase is able to traverse the terminator, leading to very, very low levels of production even when the SOS promoter is fully active. This gene is responsible for lysing the cell once it has produced large quantities of colicin, allowing the colicin out. As a note, even though this system requires the cell to commit suicide, it is still relatively efficient because it can take as little as one of the released colicins to kill another cell.

This operon is available without the SOS promoter (K131009), which is used as one of the response circuits in the University of Calgary 2008's champion cell (K131010).

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2228
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Pictures

Characterization of Colicin E2

Details of inducing colicin can be found under Part Design. Explore the University of Calgary's 2008 wiki to see how it was used.

Parameter Value and Description
Optimal Temperature 37°C
Required Bacteria RecA Positive E. coli, such as BL21
Optimal Mitomycin C Concentration Works with 1µg/mL final concentration. In a 5 mL culture, this would be 10µL of 0.5mg/mL mitomycin C.
Optimal Mitomycin C Induction Time 2-4 hours before spotting.
Lawn Characteristics 100µL of OD=0.3-0.5 (at 600nm, exponential phase), freshly plated.
Spotted Culture Supernatant (as Colicin E2 is excreted) or resuspended cells, washed with LB (to remove mitomycin C) work equally well.
[edit]
Categories
//biosafety/xnase
Parameters
None