Team:Paris Bettencourt/Suicide

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iGEM Paris Bettencourt 2012

Suicide System

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

  1. Design an anti-toxin part from the colicin E2 immunity gene that confers immunity against the colicin E2 activity protein
  2. Develop RFP labeled MG1655 Z1 strains immune to colicin E2 activity protein
  3. Measure viability of immune and vulnerable MG1655 Z1 cells in the presence of WT ColE2 cells
  4. 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
  5. Measure viability of immune and vulnerable MG1655 Z1 cells in the presence of synthetic ColE2 cells
  6. Tune the concentrations of toxin (colicin E2 activity protein)
  7. Develop RFP labeled MG1655 Z1 strains immune to toxin (colicin E2 activity protein) and anti-toxin (colicin E2 immunity protein)
  8. Combine our system with the Restriction Enzyme Group`s work, measure viability

Colicin

Colicins are the types of toxic proteins produced by strains of E.coli, and is a type of bacteriocin [1]. In natural systems the colicin operons are regulated under the SOS promoter.

In this project we tested Colicin E2 and E7, but at the end we chose to focus on Colicin E2 because it is easier to induce.

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.

PSG008.jpg PSG025.jpg

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 E.coli Cells

-MG1655.023: MG1655 cells transformed with constitutive RFP (from Anderson promoter library, BBa_J61002)

-Col E2: Wild Type Colicin E2 cells, containing pColE2-P9 plasmid [2]

Protocol

  • Plate 10 uL of 0.1 OD lawn cells from liquid culture on plain LB plates
  • Spot 5 uL of saturated Colicin E2 cells from liquid culture on plates
  • Incubate plates overnight

Results

ParisB SG assay.jpg

Characterization of the Immunity Plasmid

To test the function of our immunity plasmid, we performed Colicin E2 toxicity assays on cells transformed with our plasmid. Here we expect zones of inhibition (ZOI, clear rings around the colicin cells) when ColE2 is spotted on vulnerable cells, and the absence of ZOI in the transformed immune 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, containing pColE2-P9 plasmid [2]

Protocol

  • Grow cells in liquid culture containing the appropriate antibiotic
  • Add IPTG (0.1mM) to appropriate liquid cultures after 2 hours (OD ~0.2)
  • Wash cells containing antibiotics/IPTG and re-suspend in plain LB
  • Plate 10 uL of 0.1 OD lawn cells from liquid culture on plain LB plates or IPTG plates
  • Spot 5 uL of saturated Colicin E2 cells from liquid culture on plates
  • Incubate plates overnight

Results

SGassay2IMM.jpg
SGassay2ctr.jpg

Testing of the system

Experimental setup

Describe the experiment

Results

Present your results

Related Parts and Links

  • BBa_K131000 Colicin E2 operon, designed by Kevin McLeod, group: iGEM08_Calgary_Wetware (2008-07-22)
  • BBa_K117001 Colicin E7 with immunity, designed by Nguyen Xuan Hung, group: iGEM08_NTU-Singapore (2008-10-07)
  • BBa_K117000 Lysis gene (promotes lysis in colicin-producing bacteria strain), designed by Nguyen Xuan Hung, group: iGEM08_NTU-Singapore (2008-10-07)
  • BBa_K117009 colicin E7 production system induced by lactose, designed by Nguyen Xuan Hung, group: iGEM08_NTU-Singapore (2008-10-08)
  • BBa_R0040 TetR repressible promoter, designed by Designed by June Rhee, Connie Tao, Ty Thomson, Louis Waldman, group: Registry (2003-01-31)
  • BBa_R0011 Promoter (lacI regulated, lambda pL hybrid), designed by Neelaksh Varshney, Grace Kenney, Daniel Shen, Samantha Sutton, group: Registry (2003-01-31)
  • BBa_R0011:Experience/iGEM10 Kyoto pLac model

References

  1. Cascales E, et al. (2007) Colicin biology. Microbiol Mol Biol Rev71:158–229.
  2. Majeed G, Gillor O, Kerr B, Riley MA. (2011) Competitive interactions in Escherichia coli populations: the role of bacteriocins. The ISME Journal 5, 71-81.
  3. Pugsley AP. (1985) Escherichia coli K12 strains for use in the identification and characterication of colicins. J Gen Microbiol 131: 369-376.

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