Molecular biology and genetic engineering

Experiment 7

Aim of the Experiment

To prepare competent cells and transform plasmid DNA.

Introduction:

Bacterial transformation is a process which involves genetic alteration of bacteria by incorporation and stable expression of a foreign genetic material from the environment or surrounding medium. Since DNA is a very hydrophobic molecule, it will not normally pass through a bacterial cell membrane. In order to uptake the foreign DNA, the bacterial cells must first be made competent. Competence is the ability of a cell to take up extracellular DNA from its environment. There are different methods of carrying out transformation, e.g. chemical transformation, electroporation, gene gun, liposome mediated transfer and microinjection. Chemical transformation includes the usage of Calcium chloride (CaCl2). This mode of transformation is easy to perform and requires minimum number of equipment.

Principle:

For the incorporation of plasmid into a cell, bacteria must first be made “competent”. This process includes the treatment of cells with bivalent calcium ions in ice-cold condition. As a result small pores are formed on the cell membrane, which makes it permeable. The plasmid DNA may adhere to the surface of the cell and uptake is mediated by a pulsed heat shock at 42o C. A rapid chilling step on ice ensures the closure of the pores. These cells are allowed to propagate and selection of transformants can be done by growing the cells on a selective media which will allow only the plasmid containing cells to grow. Plasmids are extrachromosomal DNA element capable of independent replication inside a suitable host. Plasmids encode a wide variety of genes, including those required for antimicrobial resistance. These genes act as selective markers when a transformation experiment is carried out. The E. coli plasmid pUC19 encodes a gene that can be used as a selectable marker during a transformation experiment. pUC19 has ampicillin resistance marker that enables only transformed cells to grow on LB – Ampicillin plates. Transformants, thus having the ability to grow on ampicillin plates can be selected. This process of direct selection of recombinants is called insertional-inactivation. pUC19 also carries the N-terminal coding sequence for β-galactosidase of the lac operon. The E. coli host strain has a deletion at the amino terminal end of the LacZ gene, which codes for β-galactosidase. When pUC19 is transformed into the competent host cells, the truncated products from both complement each other and as a result enzymatically active β-galactosidase is produced. This is called α-complementation. The tranformants turn blue on X-gal and IPTG containing plates due to the production of β-galactosidase. X-gal is the chromogenic substrate of β-galactosidase and IPTG acts as the gratuitous inducer for the expression of this enzyme.

Materials required:

Glasswares: Conical flask, Measuring cylinder, Beaker

Other requirements: Micropipettes, Tips, 50 ml Centrifuge Tubes, Water bath (42o C), 37o C Incubator, 37o C Shaker, Centrifuge, UV Transilluminator, Crushed ice, Sterile double distilled water, Sterile loop and spreader.

Procedure:

Day 1:

1. Open the vial containing culture and resuspend the pellet with 0.25 ml of LB broth.

2. Pick up a loopful of culture and streak onto LB agar plate.

3. Incubate overnight at 370 C. Streak a loopful of culture from the stab E. coli Host) on to LB Agar plate.

Day 2:

1. Inoculate a single colony from the revived plate in 1 ml LB broth.

2. Incubate at 37o C overnight.

Day 3:

1. Take 50 ml of LB broth in a sterile flask. Transfer 1 ml of overnight grown culture into this flask.

2. Incubate at 37o C shaker at 300 rpm for 3-4 hours till the O.D600 reaches ~ 0.6.

• A) Preparation of Competent Cells:

Note: Prepare competent cells within 3 days of reviving the strain.

1. Transfer the above culture into a prechilled 50 ml polypropylene tube (not provided).

2. Allow the culture to cool down to 4o C by storing on ice for 10 minutes.

3. Centrifuge at 5000 rpm for 10 minutes at 4o C.

4. Decant the medium completely. No traces of medium should be left.

5. Resuspend the cell pellet in 30 ml prechilled sterile 0.1 M Calcium chloride solution.

6. Incubate on ice for 30 minutes.

7. Centrifuge at 5000 rpm for 10 minutes at 4o C.

8. Decant the calcium chloride solution completely. No traces of solution should be left.

9. Resuspend the pellet in 2 ml prechilled sterile 0.1M Calcium chloride solution.

10. This cell suspension contains competent cells and can be used for transformation.

• B) Transformation of cells:

1. Take 200 ul of the above cell suspension in two 2.0 ml collection tubes and label them as ‘control’ and ‘transformed’. Add 2 l of plasmid DNA to the tube labeled as transformed and mix well.

2. Incubate both the tubes on ice for 30 minutes.

3. Transfer them to a preheated water bath set at a temperature of 42o C for 2 minutes (heat shock).

4. Rapidly transfer the tubes to ice-bath. Allow the cells to chill for 5 minutes.

5. Add 800 ul of LB Broth to both the tubes. Incubate the tubes for 1 hour at 37o C to allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid.

6. Take four LB agar plates containing ampicillin, X-Gal, IPTG and label them as control, A, B and C. Plate 200 ul of culture from the ‘control’ tube and plate it on the corresponding plate with a sterile spreader. Plate 50 ul, 100 ul and 200 ul of cell cultures from the ‘transformed’ tube on to the plates labeled as A, B and C, respectively.

7. Store at room temperature till the plates are dry.

8. Incubate the plates overnight at 37o C.

Observation and Result:

After incubation, observe the plates for bacterial growth and count the number of visible colonies. Calculate the efficiency of transformation.

Calculation of transformation efficiency: Transformation efficiency is defined as the number of cells transformed per microgram of supercoiled plasmid DNA in a transformation reaction.

Transformation Efficiency = Number of colonies x 1000 ng/ Amount of DNA plated (ng) = cells/µg

Concentration of plasmid DNA provided - 80-100 ng/microlitre.

Questions:

1. What are the possible reasons of transformants are few or not blue in color?

2. How could you justify contamination observed on the plate?

3. what are the possible reasons of few or no colonies in the plate?

4. What is bacterial transformation?

5. Explain the primary physical obstacle to transforming bacteria in an experimental setting.

6. What is the purpose of exposing the bacterial cell and plasmid mixture to cold and then hot temperatures?

Reference Material

T.A. Brown - Cloning.

https://himedialabs.com/TD/HTBM017.pdf