Part:BBa_K914001:Experience

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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 (ZOI) when Col E2 is spotted on vulnerable cells, and the absence of ZOI in the transformed immune cells.

Experimental setup

Cell Types

• MG1655: These cells approximate wild type E.coli cells as they have undergone minimal genetic manipulation as compared to other laboratory strains. Genotype: F- λ- ilvG- rfb-50 rph-1
• MG1655.RFP: 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.Imm: NEB turbo cells transformed with the anti-toxin plasmid (BBa_K914001 inside pSB3C5). This strain is commonly used in the lab as it grows rapidly (visible colonies on agar, ~6.5 hours; shaking liquid culture OD 600 = 2.0, ~4 hours). This strain is also T1 phage resistant and importantly it expresses the Lac repressor (lacR), which allows us to induce the production of the immunity protein the pLac promoter with IPTG. Genotype: F´ proA+B+ lacIq ∆ lacZ M15/ fhuA2 ∆(lac-proAB) glnV gal R(zgb-210::Tn10)TetS endA1 thi-1 ∆(hsdS-mcrB)5

References:New England Biolabs, product catalogue number C2984H

• Z1.Imm: MG1655 Z1 cells transformed with the anti-toxin plasmid (BBa_K914001 inside pSB3C5). MG1655 Z1 contain the Z1 cassette (lacR tetR SpR), which allows us to induce the production of the immunity protein under control of the pLac promoter with IPTG.

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.

Quantitative Characterization of the Anti-toxin Plasmid

We characterized the plasmid pLac + ColE2 immunity gene (BBa_K914001 inside pSB3C5) by testing the response of the cells when we mix them with Colicin producing cellsin various concentration with also various concentration of IPTG as the inducer of pLac. We counted the number of the cells survived in the tests to see the effectiveness of the immunity protein in protecting the cells from Colicin toxic.

Protocol

1. Grow two overnight cultures of NEB Turbo cells containing for each culture BBa_K914001 and 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.

	100uM IPTG	10uM IPTG	1uM IPTG	0 IPTG	pSB3C5 (-) control
1x Colicin	94	79	73	42	0
1000x Colicin	9	24	26	74	0
No Colicin (+) control	36	28	30	18	27

In this experiment we expected to see more survival if we induce the cells with more IPTG and/or mix them with less Colicin. The results showed that the immunity protein can indeed protect the cells from saturated colicin, but the Lac promoter is significantly leaky. Also we noticed that the survival on the positive controls (mixture without Colicin) was somehow less than the one with Colicin. We guessed that the problems are

• The plates have more cells when they have more Colicin: the Colicin cells maybe took the Immunity/Cm-resistance plasmid
• Less cells on the plate without Colicin (and very few cells overall compared to the theoretical calculation): 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).

Therefore we still need to improve protocol to provide a more reliable quantitative data.

Perspective

For future experiment, instead of mixing cells with Colicin cells, we could 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. Anyway we have tried to make Colicin supernatant from heat shocked ColE2 cells of OD 1.0 but it failed to kill even the control cells without immunity. We suggest in the future we could give stronger stress to the Colicin to induce more the SOS promoter; such as UV light.

User Reviews

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•••• aprastowo/jrieders

The results indicate that the 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.

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