Team:Paris Bettencourt/Suicide

From 2012.igem.org

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(Mixing Immune Cells with Colicin protein)
(Mixing Immune Cells with Colicin protein)
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====Mixing Immune Cells with Colicin protein====
====Mixing Immune Cells with Colicin protein====
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'''Preparation of the immunity cells'''
'''Preparation of the immunity cells'''
#Grow overnight culture of pSG.008 (pLac-Immunity) and pSG.001 (pSB3C5) with antibiotic (Cm).
#Grow overnight culture of pSG.008 (pLac-Immunity) and pSG.001 (pSB3C5) with antibiotic (Cm).
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#Put IPTG with different concentration in each tube (pSG.008: 100uL, 10uL, 1uL and 0/no IPTG).
#Put IPTG with different concentration in each tube (pSG.008: 100uL, 10uL, 1uL and 0/no IPTG).
#Incubate again until the OD reaches 1.0.
#Incubate again until the OD reaches 1.0.
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'''Preparation of the colicin supernatant'''
'''Preparation of the colicin supernatant'''
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#Incubate in ice for 30 minutes, heat shock 42C for 30 seconds and put back in ice for 2 minutes.
#Incubate in ice for 30 minutes, heat shock 42C for 30 seconds and put back in ice for 2 minutes.
#Centrifuge the cells and collect the supernatant.
#Centrifuge the cells and collect the supernatant.
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'''Experiment'''
'''Experiment'''

Revision as of 01:26, 25 September 2012


iGEM Paris Bettencourt 2012

Suicide System

Contents

Overview

Our goal is to engineer a synthetic toxin-anti-toxin system from the wild type Colicin E2 (Col E2). This synthetic toxin-anti-toxin system is receptor specific, allows for delayed population-level suicide, complete genome degradation, and will function on a tunable delay.

Background

What are Colicins? Colicins are proteins produced by some types of E.coli that are toxic for sensitive species of related E.coli. Colicinogenic cells contain specific immunity against their own toxin, and their colicins only effect cells containing specific receptors on their outer membrane surface. Colicins are therefore classified by the receptor to which they bind to, for example, colicins E1-E9 bind to the outer membrane (OM) protein BtuB which mediates the entry of nucleosides, siderphores, and vitamin B12 in the cell. Colicins E1-E9 are also Group A colicins, meaning that they are translocated through the cell envelope by the Tol machinery, whereas Group B colicins are translocated through the cell envelope by the TonB machinery. Colicins have different modes of action, including enzymatic activity such as DNase (DNA cleaving) activity, and pore-forming activity. Colicins have three domains, the N-terminal domain functions in translocation through the membrane, the central domain is involved in binding to the OM receptor, and the C-terminal domain contains active (lethal) protein region [1].

Colicinbiofig3.jpg

Image taken from Cascales E, et al. (2007) Colicin biology. Microbiol Mol Biol Rev 71:158–229

For our project we have selected Colicin E2, which has enzymatic DNase activity. Colicin E2 is ideal because it not only kills sensitive cells on a population level, but also destroys genomic material, preventing the spread of genetically modified parts via horizontal gene transfer.

In natural systems colicin operons are regulated under the SOS promoter (Stress response). Group A colicins contain type I plasmids. These plasmids are generally 6-10 kb, found in about 20 copies per cell, and can be amplified and mobilized in the presence of a conjugative plasmid. The first gene of the colicin operon is the activity protein, named Colicin X activity (i.e. cea for Colicin E2) . This is followed by the immunity protein, named Colicin X immunity (cei), which, like the activity protein, is regulated by the LexA promoter, but also has its own constitutive promoter. This separate promoter is located with in the coding sequence of the cea and allows for constant overproduction of the cei, thereby protecting the producing cell. The last gene codes for the lysis protein, named colicin X lysis protein (cel), which causes the release of colicin into medium and is responsible for the cell death of the producer. The SOS promoter can be induced by UV light, chemicals, and stress conditions.

ColE2P9.jpg

We would like to clone the cea and cei of Colicin E2 onto two separate plasmids, creating toxin and anti-toxin plasmids.

Bigger Picture

Our anti-toxin plasmid can be combined with the restriction enzyme system to generate a toxin-anti-toxin system that works on a tunable delay depending on the induction of the restriction enzyme. Once the degradation of the anti-toxin plasmid containing the immunity protein is initiated, the cell will continue to produce the activity protein, which will digest the cells own genetic material as well as its neighbors and extracellular DNA via its DNase activity.

Remove the Restriction module of the scheme.

File:Paris Bettencourt 2012 General Circuit s1.gif

Objectives

In this project, our goal is to construct a well characterized toxin-antitoxin system using the Colicin system (design) by:

  1. Design an anti-toxin part from the Col E2 immunity gene that confers immunity against the Col E2 activity protein
  2. Measure viability of immune and vulnerable NEB turbo and MG1655 Z1 cells in the presence of WT Col E2 cells
  3. Design a Col E2 activity protein part under the control of a constitutive promoter and establish in immune NEB turbo and MG1655 Z1 cells to generate synthetic Col E2 cells
  4. Measure viability of immune and vulnerable NEB turbo and MG1655 Z1 cells in the presence of synthetic Col E2 cells
  5. Combine our system with the Restriction Enzyme System, measure viability

Design

For our system we will generate two plasmids, one carrying the Col E2 activity protein and one carrying the Col E2 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, to drive the expression of the activity protein so that it would not overwhelm the cells until after the desired delay. We ultimately plan to integrate the activity protein cassette into the genome of our bacteria.

PSG008.jpg

We do not require the colicin lysis protein for our system, and while the activity protein may degrade extracellular DNA released from our genetically modified bacteria, it is preferable that the cells do not lyse and the genomic material is digested within the cells. A small percentage of cells will die naturally and release the activity protein, which will have a lethal action on any cells that escape our system via mutation of the activity protein gene.

Experiments and results

Characterization of the Colicin E2 Toxicity

We performed a basic assay to characterize the toxicity of the Col E2 cells. Here we expect zones of inhibition (ZOI, clear rings around the colicin cells) when Col E2 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

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

Results

Col E2 cells produce ZOI in sensitive cells, as indicated bellow by red arrows. MG1655 and MG1655.023, cells which are not colicinogenic, do not produce ZOI when spotted on a lawn of sensitive cells.

ParisB SG assay.jpg

Interpretation

The Col E2 cells secrete the lethal activity protein, which diffuses out from the Col E2 colonies spotted on the LB plates. This generates a region where vulnerable cells can not grow, visible as an empty ring (ZOI) around the Col E2 colonies. It is important to note that Col E2 cells do not need to be induced to produce sufficient activity protein to generate the ZOI. These results confirm the toxicity of Col E2 cells against MG1655 and MG1655.023, related species of E.coli. We also assayed Top10 cells, NEB turbo cells, MAGE cells, and MG1655 ZI.023 cells, for more details look here

Characterization of the Anti-toxin Plasmid

To test the function of our anti-toxin plasmid, we performed Colicin E2 toxicity assays on cells transformed with our plasmid. Here we expect zones of inhibition when Col E2 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]
  • NEB.008: NEB turbo cells transformed with the anti-toxin plasmid (pSG.008)
  • ZI.008: MG1655 ZI cells transformed with the anti-toxin plasmid (pSG.008)

Protocol

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

Results

Col E2 cells do not generate a ZOI in cells containing the anti-toxin plasmid, in both induced and un-induced cells. However, Col E2 cells do produce a ZOI in vulnerable MG1655 cells as indicated by red arrows below.

SGassay2IMM.jpg
SGassay2ctr.jpg

Interpretation

These results indicate that our anti-toxin plasmid indeed confers immunity to sensitive cells, however, immunity is not dependent on IPTG induction. This is not completely surprising given that the immunity protein has an incredibly high affinity for the activity protein and that pLac is known to be leaky. Even a small concentration of immunity protein could protect un-induced cells against the DNase activity of the colicin activity protein.

Quantitative Characterization of the Anti-toxin Plasmid

We want to characterize the plasmid (pLac + ColE2 immunity gene) by varying the concentration of IPTG and the ColicinE2 cells.

Mixing Immune Cells with Colicin Cells

Protocol

  1. Grow overnight culture pSG.008 (pLac-Immunity) and pSG.001 (pSB3C5) with antibiotic (Cm).
  2. Centrifuge and resuspend in LB.
  3. Prepare 5 tubes of 10ml LB and put 40uL of pSG.008 cells into 4 tubes and pSG.001 cells in 1 tube.
  4. Incubate 37C until the OD reaches 0.2
  5. Put IPTG with different concentration in each tube (pSG.008: 0.1mM, 0.05mM, 0.025mM and 0/no IPTG).
  6. Incubate again until the OD reaches 1.0
  7. Take each 0.5ml and mix with 0.5ml Colicin cells (saturated, saturated diluted 1000x, plain LB with corresponding amount of IPTG) in small 2ml eppendorf tubes.
  8. Incubate for 30 minutes.
  9. Dilute 10000x (100x then 100x) in MgSO4 e-2M.
  10. Plate 20uL in Cm to kill the Colicin cells and leave the immune cells.

Note: 1ml of cells of OD 1.0 has 1e9 cells, so we expect if all cells survive we will get 1000 colonies at the end.


Results

The table shows the number of colonies survive in each plate IPTG x Colicin.

SG exp230912.png

In this experiment we had some weird things which we try to explain

  • The plates have more cells when they have more Colicin; the Colicin maybe take the Immunity/Cm-resistance plasmid
  • Less cells on the plate without Colicin; the cells may have died because they loose the Immunity/Cm-resistance plasmid during the incubation without antibiotic (which we did to avoid killing the Colicin cells)

Solution for the next experiment: instead of mixing cells with Colicin cells, we will mix them with the supernatant of centrifuged Colicin cells (only the ColE2 protein). Therefore we can put the antibiotic during the whole experiment to avoid plasmid loss.

Mixing Immune Cells with Colicin protein

Preparation of the immunity cells

  1. Grow overnight culture of pSG.008 (pLac-Immunity) and pSG.001 (pSB3C5) with antibiotic (Cm).
  2. Centrifuge and resuspend in LB.
  3. Prepare 5 tubes of 10ml LB and put 40uL of pSG.008 cells into 4 tubes and pSG.001 cells in 1 tube (negative control).
  4. Incubate 37C for 30 minutes.
  5. Put IPTG with different concentration in each tube (pSG.008: 100uL, 10uL, 1uL and 0/no IPTG).
  6. Incubate again until the OD reaches 1.0.


Preparation of the colicin supernatant

  1. Grow overnight culture of Colicin E2 cells (pColE2-P9).
  2. Put 40uL of the saturated cells into 10ml fresh LB.
  3. Incubate 37C until the OD reaches 1.0.
  4. Incubate in ice for 30 minutes, heat shock 42C for 30 seconds and put back in ice for 2 minutes.
  5. Centrifuge the cells and collect the supernatant.


Experiment

  1. Dilute the tested cells 10000x (100x then 100x) in LB with antibiotic and corresponding concentration of IPTG.
  2. Take each 0.5ml and mix with 0.5ml Colicin supernatant (undiluted, diluted 1000x, plain LB with antibiotic and corresponding amount of IPTG) in small 2ml eppendorf tubes.
  3. Incubate for 30 minutes.
  4. Plate 20uL in Cm.

Note: 1ml of cells of OD 1.0 has 1e9 cells, so we expect if all cells survive we will get 1000 colonies at the end.

Discussion

Cloning the colicin E2 activity protein, given its lethal nature, is a great challenge for us. We took several approaches. We established immune strains expressing excess immunity protein in which we could transform the toxin plasmid. We tried cloning the toxin without a promoter and finally tried various vectors with and with out promoters. Although the immune strains are viable, transformable, and prove immune to wild type Colicin E2 cells, we have yet to successfully transform a single toxin plasmid.

However, developing and characterizing the immune strains was not as straight forward as we imagined. Whether in NEB turbo or MG1655 Z1 cells, the immune strains grew incredibly slowly. Despite being under the control of pLac, their immunity proved somewhat independent of IPTG, as both un-induced and induced cells are immune to Colicin E2. In addition, we had some strange toxicity assay results, partially resolved by the explanation of Colicin E2 contamination.

To see more details on the toxicity assays, click here

We will continue on our quest to clone the colicin E2 activity protein, and hope that future teams can learn from our experience.

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 Rev 71:158–229.
  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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