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This is content one Minicells are achromosomal cells formed by aberrant cell division in living cells. Several mutant strains of E. coli (P678-54, χ925) have been isolated that have been shown to harbor a mutation in a particular set of genes. FtsZ, one of the proteins involved in cell division has been shown to be one of those. Overexpression of this protein leads to septation at the poles, leading to the formation of the so-called minicells. The non-replicative behaviour of the minicells gives rise to a broad spectrum of applications. Our project focusses on engineering minicells to produce certain novel peptides called bacteriocins. Bacteriocins are antimicrobial peptides (AMPs) produced by almost all bacteria having specific detrimental effect on the surrounding microbial community. Hence engineering minicells to produce bacteriocins can have a wide range of applications in fields like animal husbandry, human probiotics and strain selection. Minicells will be engineered as probiotics to produce bacteriocins and can be used as a defence against common infections and diseases in the gastro-intestinal tract. Because the minicells do not have the ability to multiply, there is no risk of infection associated with children and immune-compromised people. Pseudomonas aeruginosa has been selected as a model gastro-intestinal pathogen. The appropriate bacteriocin has been chosen to be Thuricin S, produced by Bacillus thuringiensis. One of the most common issues in the field of Medicine and in general, Biology, the formation of MRSA biofilms, can also be addressed with this approach. Studies have shown that a bacteriocin, Lysostaphin, can penetrate the biofilm and act on Staphylococcus aureus. But, the organism has already developed resistance towards lysostaphin. Building on that premise, we will be engineering minicells to produce Bactofencin A, a novel cationic bacteriocin produced by Staphylococcus simulans and test its functionality. Aside from the above areas, there are other prospective areas where minicells producing bacteriocins can be applied. Bacteriocins are highly specific with respect to their target organism and will selectively kill the undesired organism to which it is directed against. This property can be utilized for strain selection on petri-plates. In addition, Clostridium botulinum – specific bacteriocin producing minicells can also be engineered to prevent botulism through packaged foods.

RESTRICTION DIGESTION

MATERIALS REQUIRED

• Ice container
• Eppendorfs
• Purified DNA (>16 ng/µl)
• Double distilled water (nuclease-free)
• BSA (4 mg/ml)
• Restriction enzymes and their respective buffers (10x)

PROCEDURE

1. Add about 2 µl of the DNA to be restricted to an eppendorf placed on ice.
2. Add 2 µl of the restriction enzyme buffer.
3. Add 0.5 µl of BSA (Addition of BSA enhances performance of the restriction enzyme by providing additional protein to stabilize the enzyme and balances side-effects arising as a result of enzyme interaction with solid surfaces like the walls of the tube).
4. Add 0.5 µl of the restriction enzyme.
5. Make up the reaction volume to 20 µl with distilled water (The 10x buffer gets diluted to 1x).
6. The reaction is incubated at 37 oC for 30 minutes.
7. To denature the enzymes present in the reaction mix, the mixture is kept at 80 0C for 20 minutes and the digested fragments are visualized by agarose gel electrophoresis.

LIGATION TO PLASMID

MATERIALS REQUIRED

• Linearized plasmid backbones
• Restriction-digested fragments with complementary overhangs
• T4 DNA ligase and buffer
• Double distilled water (nuclease-free)

PROCEDURE

1. Add 2 µl (around 25 ng) of the linearized plasmid backbone to an eppendorf on ice.
2. Add equimolar amounts of restricted DNA fragments to the eppendorf.
3. Add 1 µl of T4 DNA ligase buffer.
4. Add 0.5 µl of T4 DNA ligase enzyme and make up the volume to 10 µl using double distilled water.
5. Incubate the reaction at 16 oC for 30 minutes.
6. Heat kill the enzyme at 80 oC for 20 minutes.

AGAROSE GEL ELECTROPHORESIS

• The phosphate backbone of DNA is negatively charged.
• Migrates towards the positive pole in an electric field.
• Separation during migration is based on the length of the DNA fragment, strength of the electric field and the concentration of agarose in the gel among others.
• EtBr is added as a DNA intercalating agent and fluoresces under UV light.

MATERIALS REQUIRED

• Gel tank apparatus
• Power supply
• Agarose
• 10x TBE buffer o 108 g tris
o 55 g boric acid
o 40 ml 0.5 M EDTA
o Made up to 1 l with distilled water
• EtBr (10 mg/ml)
• 6x Gel loading dye
• DNA ladder
• Sample DNA
• UV transilluminator

PROCEDURE

Preparation of agarose gel

1. Dissolve agarose in 1x TBE buffer to the required concentration (0.8% is the standard, higher or lower concentrations can be used for separating DNA fragments with very low or very high difference in their lengths respectively).
2. Heat the solution in a microwave to melt the agarose and dissolve it completely.
3. When it cools down to hand-bearable temperature, add EtBr so that the final concentration in the gel is about 0.3-0.5 µg/ml.
4. Pour the gel onto a leak-proofed case with a comb inserted and allow the gel to solidify (The gel turns from completely transparent to cloudy after it solidifies).

Loading and running

1. Mix the DNA with the loading dye (1x) (1:1) on a piece of parafilm stuck to the work bench.
2. Load the mixture onto the gel.
3. Load the DNA ladder at the ends of the gel (if the length of the sequence needs to be determined).
4. Run the gel at 60-90 V for as long as required (60-90 minutes should be sufficient enough).
5. View the resolved DNA bands under the UV transilluminator.

ELUTION OF DNA FROM AGAROSE GELS

MATERIALS REQUIRED

• Elution buffer (0.5 M sodium acetate (pH 7) and 1 mM EDTA (pH 8))
• 95% ethanol
• 80% ethanol
• TE buffer (10x) (pH 8.0)
o 10 mM tris
o 1 mM EDTA
• Gel fragment containing the DNA

PROCEDURE

1. Excise the region of the gel containing the required DNA band using a scalpel.
2. Add elution buffer to the gel slice until the level of the buffer is a few mm above the level of the excised gel band.
3. Heat the solution in a water bath at 65 oC until the agarose completely melts.
4. Fast-freeze by placing in a -80 oC freezer for 10-15 minutes.
5. Immediately thaw the solution by centrifuging for 10 minutes.
6. The supernatant is transferred to a new tube.
7. Add elution buffer again to the pellet and repeat the steps 3-6.
8. Accumulate the supernatants and add an equal volume of 1-butanol.
9. Rock the mixture for 15 minutes to remove the EtBr completely from the gel.
10. Discard the supernatant and repeat the steps 8 and 9 2-3 times.
11. Add 2.5 volumes of cold 95% ethanol and mix. Precipitate the mixture at -70 oC for atleast 30 minutes.
12. Centrifuge for 15 minutes and decant off the supernatant.
13. Add 200 µl of cold 80% ethanol to the pellet and mix until the pellet is dislodged from the bottom of the tube.
14. Centrifuge for 5 minutes and discard the supernatant.
15. Dry the DNA pellet and dissolve it in 20 µl of 0.5x TE buffer.

PREPARATION OF COMPETENT CELLS

• Competence is the ability of a cell to take up extrachromosomal DNA
• Rapidly dividing cells can be made competent to take up plasmid DNA by treatment with CaCl2
• CaCl2 interacts with the host cell membrane and makes the membrane more permeable to extracellular plasmid DNA.

MATERIALS REQUIRED

• E.coli cells (mostly DH5α is used)
• SOB medium
o 2% w/v tryptone
o 0.5% w/v yeast extract o 8.56 mM NaCl
o 2.5 mM KCl
o 10 mM MgCl2
o 10 mM MgSO4
• CCMB80 buffer (pH 6.4)
o 10 mM potassium acetate (pH 7)
o 80 mM CaCl2.2H2O o 20 mM MnCl2.2H2O o 10 mM MgCl2.2H2O o 10% glycerol

PROCEDURE

Preparation of seed stocks

1. Inoculate single colonies of E.coli to 2 ml of SOB medium and shake overnight.
2. Add glycerol to 15%.
3. Aliquot 1 ml samples and store at -80 oC.

Preparation of competent cells

1. Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20 oC to an OD of 0.3.
2. Pellet the cells by centrifuging at 3000 xg for 10 minutes at 4 oC.
3. Resuspend the cells in 80 ml of cold CCMB80 buffer.
4. Incubate on ice for 10 minutes.
5. Centrifuge at 3000 xg for 10 minutes at 4 oC.
6. Resuspend the cell pellet in 10 ml of ice cold CCMB80 buffer.
7. Aliquot 50 µl samples and store them at -80 oC.

ISOLATION OF PLASMID DNA

MATERIALS REQUIRED

• Plasmid DNA - containing cell culture
• P1 buffer (pH 8.0) (store at 4 oC) o 50 mM tris-Cl pH 8.0
o 10 mM EDTA
o 100 µg/ml RNaseA
• P2 buffer
o 200 mM NaOH
o 1% (w/v) SDS
• N3 buffer (pH 4.8)
o 4.2 M Guanidium hydrochloride
o 0.9 M Potassium acetate
• Isopropanol
• 70% ethanol
• 0.5x TE buffer

PROCEDURE

1. Pellet the cells by centrifuging them at 4000 rpm for 10 minutes
2. Resuspend the pellet in 250 µl of P1 buffer and vortex.
3. Add 250 µl of P2 buffer and mix by inverting the tube (the solution should turn clear).
4. Add 350 µl of N3 buffer and immediately mix the tube by inverting and avoid localized precipitations (the solution should become cloudy once again).
5. Centrifuge for 10 minutes at 13000 rpm.
6. Transfer the supernatant to a fresh tube.
7. Add around 500 µl of isopropanol (a cloudy precipitate should form).
8. Centrifuge at 8000 rpm for 10 minutes.
9. Discard the supernatant and wash the pellet atleast twice using 70% ethanol.
10. Air-dry the tubes and dissolve the pellet in 20 µl of 0.5x TE buffer.
11. Store the tubes at -20oC.

TRANSFORMATION

MATERIALS REQUIRED

• Competent cells
• Plasmid DNA
• Ice box
• Boiling water bath
• SOC medium
o Add 20 ml of filter-sterilized 20% glucose solution to 1 l of SOB medium

PROCEDURE

1. Thaw the competent cells on ice for about 15-20 minutes.
2. Pipette 50 µl of competent cells into a 2 ml tube placed on ice.
3. Add 1 µl of DNA to the tube.
4. Mix well and incubate the tubes on ice for 30 minutes.
5. Provide heat shock by placing the cells at 42 oC for 1 minute and immediately transfer the tubes to ice and incubate or 5 minutes.
6. Add 200 µl of SOC medium to each tube and incubate at 37 oC for 2 minutes.
7. Spread plate 20 µl of the culture onto the appropriate antibiotic - containing plates.
8. Incubate the plates overnight and check for the transformation efficiency.

PREPARATION OF ANTIBIOTIC STOCKS

AMPICILLIN

Stock - 100 mg/ml in 50% ethanol

Working - 100 µg/ml

Ampicillin is light sensitive. Store at 4 oC protected from light.

KANAMYCIN
Stock - 35 mg/ml in distilled water

Working - 35 µg/ml

Kanamycin is light sensitive. Store at 4 oC protected from light.

CHLORAMPHENICOL
Stock - 35 mg/ml in 100% ethanol

Working - 35 µg/ml

TETRACYCLINE
Stock - 15 mg/ml in 50% ethanol

Working - 15 µg/ml

Tetracycline is light sensitive. Store at 4 oC protected from light.

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