BACTERIAL TRANSFORMATION :

AIM :

To transform competent E. coli strains using plasmid DNA.

THEORETICAL BACKGROUND :

Bacterial transformation is a process by which a recipient cell acquires genes from a free DNA molecule in surroundingmedium. Earlier transformation was one of the most important mapping techniques and at present too it remains the only means of genetic mapping for certain organisms.

In 1928, Frederick Griffith carried out an elegant experiment which led to the demonstration that DNA is the genetic material. He conducted his experiments using Streptococcus pneumoniae R and S strains (R-rough type colonies which are non-pathogenic and S-smooth type colonies which are pathogenic due to the synthesis of capsular polysaccharide) which are injected into mice. Mice injected with R- pneumococci or with heat killed S-cells remains remainedhealthy, but mice injected with a mixture consisting of a small number of R -bacteria and a large number of heat killed S-cells died of pneumonia. Several more sophisticated experiments have led to conclusion that substance responsible for genetic transformation was DNA of donor cells and hence DNA was proved to be genetic.

 

Transformation begins with uptake of DNA fragment from the surrounding medium by a recipient cell and terminates with recombinant exchange of part of DNA fromdonor with homologous segment of recipient chromosome. DNA is a very hydrophobic molecule and will not normally pass through a bacterial cell membrane. Since ability of cells to uptake DNA is limited, certain conditions are created so that uptake is greatly enhanced. A population such cells are called 'competent cells'.

Competence is the ability of a cell to take up extracellular DNA from its environment. It is a definite physiological state and hence can be induced by certain growth factors. The usual technique involves a shift down in which cells are transferred from a relatively rich medium to a nutritionally poor medium resulting in appearance of variable number of competent cells in culture.

Treating bacteria with ice-cold conditions with bivalent calcium ions and briefly heating them also induces cells into a transient competent state during which recipient bacteria can take up DNA's derived from a variety of sources. The plasmid DNA may adhere to the surface of the cell and uptake is mediated by a pulsed heat shock at 42°C. Due to the heat treatment small pores are formed on the cell membrane, which makes it permeable. A rapid chilling step on ice ensures the closure of the pores.

Competence is seen to arrive at a specific stage of growth of culture i.e. late log phase and depends on culture conditions. Initially a small fraction of ceils becomes competent and excretes one or more proteins called competence factor, which converts remainder of cells into a competent one. The development of competence may require only a few minutes and can be maintained for some time. The appearance of competence can be enhanced or antagonized by presence or absence of certain amino acids such as arginine, glutamate depending upon growth conditions.

Competent cells undergo lots of changes in both cel! wall and cell membrane. The cell wall becomes more porous resulting in leakage of enzymes into the medium. There is an increase of autolytic enzyme activity within competent cells and increase in DNA degrading activity at cell surface. A change in surface charge of cell has been suggested due to binding of competence factor which is positively charged. The positive charge should make it easier for the negatively charged DNA molecule to bind to the cell surface. Ultra structural changes such as increase in the mesosome number occur in competent cells. These structures have an effect of bringing cell surface closer to centre of cells, the location of nucleoid. It has been suggested that mesosomes represent the transport mechanisms of transforming DNA fragment. Thus in an intact cell DNA does not bind preferentially to middle or tip of cell rather than distributing it randomly over the cell surface.

DNA uptake is not species specific i.e., foreign DNA from any source can be taken up by recipient cell. Example plasmid DNA may be used in transformation study.

Few bacteria such as K. pneumoniae, H. influenza are able to get transformed in nature. Mandel and Higa (1970) first showed that the treatment of bacterial cells with CaCl₂ enhances uptake of λ phage DNA. In 1973, Cohen Chan, Hsu's method was also shown to work for plasmid DNA. Once inside the bacterial cells (the phage or plasmid) DNA replicates and express markers such as antibiotic resistance that allow selection of transformed cells, conferring their survival in presence of the antibiotic.

The ability of bacteria to take up DNA is short lived. After exposure to agents that enhance uptake, most strains of bacteria remain in a competent state for only 1-2 days. Transformed 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.

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-Ampicil!in 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 p-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 inducer for the expression of this enzyme.

 

Thus, on transformation of cells with pUC19 plasmid, antibiotic resistance is conferred on the host as this plasmid carries gene for ampicillin resistance. As a result, those cells that grow in presence of ampicillin are transformed cells. The transformed colonies are blue on X-Gal, IPTG plates due to a-complementation.

General Overview.

REQUIREMENTS :

Ampicillin (50 mg/ml), Luria-Bertani (LB) broth and agar, E. coli host, plasmid DNA (50 ng/μl), chilled 0.1M sterile calcium chloride solution, X-Gal stock solution, IPTG stock solution, polypropylene 2 ml collection tubes, conical flask, measuring cylinder, beaker, micropipettes, micropipette tips, 50 ml centrifuge tubes, waterbath (42°C), incubator at 37°C, shaker at 37°C, centrifuge, UV transilluminator, crushed ice, sterile double distilled water, sterile loop and spreader.

PROCEDURE :

 

Note : The procedure described below is based on the HiPer® Transformation

Teaching Kit from Hi-Media (Product Code: HTBM017).

I. Day 1 :

a. Open the bottle containing culture and resuspend the pellet with 0.25 ml of LB broth.

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

c. Incubate overnight at 37°C.

II. Day 2 :

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

b. Incubate at 37°C overnight.

III. Day 3 :

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

b. Incubate at 37°C shaker at 300 rpm for 3-4 hours til! the Aeoo reaches ~ 0.6.

Part A : Preparation of Competent Ceils :

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

i. Transfer the above culture into a pre-chilled 50 ml polypropylene tube.

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

iii. Centrifuge at 5000 rpm for 10 minutes at 4°C.

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

v. Resuspend the cell pellet in 30ml pre-chilledsterile 0.1MCaCl2 solution.

vi. Incubate on ice for 30 minutes.

vii. Centrifuge at 5000 rpm for 10 minutes at 4°C.

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

ix. Resuspend the pellet in 2 ml pre-chilled sterile 0.1M CaCL solution.

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

Part B : Transformation of cells :

i. Take 200 μl of the above cell suspension in two 2.0 ml collection tubes and label them as 'control' and 'transformed'. Add 2 μl of plasmid DNA to the tube labeled as transformed and mix well.

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

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

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

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

vi. Take four LB agar plates containing ampiciilin, X-Gal, IPTG and label them as control, A, B and C. Plate 200 μl of culture from the 'control' tube and piate it on the corresponding plate with a sterile spreader. Plate 50 μl, 100 μl and 200 μl of cell cultures from the 'transformed' tube on the plates labeled as A, B and C, respectively.

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

viii.Incubate the plates overnight at 37°C.

IV. Observation

After incubation observe the plates for the bacterial growth and count the number of visible colonies. Calculate the efficiency of transformation. Record your observations as follows :

Denote + when you observe bacterial growth, - when there is no growth

V. 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

Notes :

1. Prior to the preparation of competent cells, pre-chill the tubes, 0.1M Calcium chloride solution and centrifuge tubes. Set the centrifuge at 4°C and water bath at 42°C.

2. Transformation should be carried out as soon as possible after the competent cells are prepared. Storage of competent cells leads to poor or no transformants.

3. Preparation of LB agar place for selection of recombinants : To 100 ml of sterile, melted LB agar cooled to 45-50°C, add 100 μl of ampicillin stock, 100 μl of IPTG and 200 μl of X-Gai. Mix well and pour into sterile Petri dishes.