Team:Paris Bettencourt/Suicide
From 2012.igem.org
Contents |
Overview
Our goal is to engineer a synthetic toxin-antitoxin system from the wild type colicin E2. This synthetic toxin-antitoxin system is receptor specific, allows for delayed population-level suicide, complete genome degradation, and will function on a tunable delay.
Objectives
- Design an anti-toxin part from the colicin E2 immunity gene that confers immunity against the colicin E2 activity protein
- Develop RFP labeled MG1655 Z1 strains immune to colicin E2 activity protein
- Measure viability of immune and vulnerable MG1655 Z1 cells in the presence of WT ColE2 cells
- Design a colicin E2 activity protein part under the control of a constitutive promoter and establish in immune MG1655 Z1 cells to generate synthetic ColE2 cells
- Measure viability of immune and vulnerable MG1655 Z1 cells in the presence of synthetic ColE2 cells
- Tune the concentrations of toxin (colicin E2 activity protein)
- Develop RFP labeled MG1655 Z1 strains immune to toxin (colicin E2 activity protein) and anti-toxin (colicin E2 immunity protein)
- Combine our system with the Restriction Enzyme Group`s work, measure viability
Design
For our system we require two plasmids, one carrying the ColE2 activity protein and one carrying the ColE2 immunity protein. As the immunity protein is normally present in excess in natural colicinogenic cells, we chose a 10-12 copy vector pSB3C5 to carry the immunity protein, and a lower ~5 copy vector pSB4K5 to carry the toxin protein. We used pLac to drive expression of the immunity protein, however, different inducible promoters should be used depending on the overall design and application of the safety circuit. We chose the constitutive promoter BBa_J23108 from the Anderson Promoter Collection, which has a relatively moderate expression level, so that the activity protein would not overwhelm the cells until after the desired delay.
Experiments and results
Characterization of the Colicin E2 Toxicity
We performed a basic assay to characterize the toxicity of the Colicin E2 cells. Here we expect zones of inhibition (clear rings around the colicin cells) when ColE2 is spotted on vulnerable cells.
Experimental setup
Cell Types:
-MG1655: Wild Type Cells
-MG1655.023: MG1655 cells transformed with constitutive RFP (from Anderson promoter library, BBa_J61002)
-Col E2: Wild Type Colicin E2 cells
Protocol
- 10 uL of 0.1 OD lawn cells from liquid culture plated on plain LB plates
- 5 uL of saturated Colicin E2 cells from liquid culture spotted on plates
- Incubate plates overnight
Results
Testing of the system
Experimental setup
Describe the experiment
Results
Present your results