Molecular Biology and genetic engineering

Experiment 6

Aim of the Experiment

Making competent cells.

Introduction:

The process of calcium chloride heat-shock transformation encourages bacterial cells to uptake DNA from the surrounding environment. The exact mechanism of how this process works is still largely unknown, but there are hypotheses on the different aspects of the procedure. The role of calcium ions in the cell suspension is hypothesized to be a cation bridge between the negative charges on phosphorylated lipid A in lipopolysaccharide (LPS), and the phosphate backbone of DNA (1, 2). The ice-cold CaCl2 solution facilitates binding of DNA to the surface of the cell, which then enters the cell after a short period of heat-shock (3). Cells that are successfully transformed are usually identified by selection or screening markers such as drug resistance or fluorescence (4). This technique is commonly used to transform cells with plasmids for various purposes like recombinant protein expression, cloning, and long term storage of the plasmids.

Principle:

Calcium chloride heat-shock transformation is a powerful molecular biology technique used to introduce foreign DNA into a host cell. The concept of the technique is to render cells competent using CaCl2 to allow for introduction of plasmid. Plasmids usually contain the gene(s) of interest in addition to selection and/or antibiotic resistance markers. The Hanahan or calcium chloride method is used to generate chemically competent cells. Heat-shocking facilitates the transport of plasmid into the competent cell. Transformed cells will allow for downstream applications such as plasmid amplification or protein expression. This methods paper will outline the protocol for the preparation of calcium competent Escherichia coli using the Hanahan method and heat-shock transformation of calcium competent Escherichia coli.

Material required:

• Competent cell preparation:

1. 1mL of overnight Escherichia coli (E. coli) culture

2. 100mL of 0.1M CaCl2 (ice cold)

3. 20mL of 0.1M CaCl2 with 15% glycerol solution (ice cold)

4. 100mL of fresh lysogeny broth (LB) media

• Heat-shock transformation:

1. 1pg - 100ng plasmid DNA (1-5uL)

2. 1mL of pre-warmed LB media or SOC media at 37C

3. LB agar plates (with appropriate reagent for selective or screening)

4. Ice

• Equipment:

1. 37C shaking incubator

2. 42C water bath

3. Spectrophotometer

Procedure:

A. CaCl2 Buffers Preparation

• 1M CaCl2 (stock solution, 10x working concentration)

1. Weigh out 11.1g of anhydrous CaCl2

2. Add to 80mL of dH2O

3. Mix solution until CaCl2 is fully dissolved

4. Top up to 100mL

5. Filter sterilize through a 0.22μm pore

• 0.1M CaCl2 (working solution)

1. Add 10mL of 1M CaCl2 to 90mL of dH2O for a 1:10 dilution

2. Filter sterilize through a 0.22μm pore

• 0.1M CaCl2 + 15% glycerol (working solution)

1. Mix 6mL 1M CaCl2 with 9mL sterile glycerol and 45mL dH2O

B. Overnight Culture(s)

1. Inoculate 1mL of LB with E. coli

2. Place in shaking incubator at 37°C and 200rpm

3. Incubate for 12-16 hours

C. Generation of Competent Cells (CaCl2 wash)

Subculturing overnight culture:

1. Add 1mL of overnight culture to 99mL of fresh LB (1:100 dilution, no antibiotics)

2. Shake incubate at 37°C and 200rpm for 3-4 hours or until OD reaches 0.4

• CaCl2 wash:

1. Ensure that all reagents (CaCl2 solutions, Oakridge tubes, centrifuge) are ice-cold or at 4°C

2. Separate culture into multiple Oakridge tubes

3. Place on ice for 20 minutes

4. Centrifuge at 4°C at 4000rpm for 10 minutes

5. Discard the supernatant by tipping tubes over a discard bin and then aspirating any remaining media.

6. Resuspend each pellet with 20mL ice-cold 0.1M CaCl2, incubate on ice for 30 minutes

7. Centrifuge at 4°C at 4000rpm for 10 minutes

8. Discard the supernatant and combine pellets by resuspending in 5mL ice-cold 0.1M CaCl2 with 15% glycerol

9. Use for downstream transformation or store in -80°C freezer

• D. Heat-shock transformation Heat-shock:

1. Thaw competent cells on ice

2. Add 1-5μl (10pg-100ng) of plasmid (do not exceed 5μL for a 50μL cell aliquot)

3. Incubate on ice for 30 minutes

4. Heat-shock by placing in 42°C water bath for exactly 30 seconds

5. Place cells on ice for 2 minutes

6. Add 1mL pre-warmed LB or SOC medium

7. Shake incubate 37oC, 200rpm, 1 hour for outgrowth

• Plating and incubation:

1. Spread plate 1:10 and 1:100 dilutions of the outgrowth cultures on warm selective and/or screening plates (e.g. Ampicillin and/or X-gal if required)

2. Incubate at 37°C for 12-16 hours

• Plate observations: Inspect plates for isolated colonies.

Observation and Result:

Anticipated results:

1. • No colonies appear on the plate.

2. • A countable number of colonies (30-300 colonies) appear on the plate.

3. • Many colonies that are too numerous to count appear on the plate.

Controls:

1. Use a DNA preparation that has been shown to give transformants in previous experiments to act as a positive control.

2. Perform another transformation to which plasmid DNA is not added to act as a negative control.

3. Transform 1ng of target plasmid to check competent cell viability, calculate transformation efficiency, and verify the antibiotic resistance of the plasmid.