Preparation of competent cells and transformation of E.coli

/Preparation of competent cells and transformation of E.coli
Preparation of competent cells and transformation of E.coli 2018-02-24T05:18:44+00:00

Preparation of competent cells and transformation of E.coli

Preparation of competent cells and transformation of E.coli

Introduction

Nucleic acids do not enter bacteria under their own power, but require assistance traversing the outer and inner cell membranes and in reaching an intracellular site where they can be expressed and replicated. Chemical methods can be used to make the cells competent and transform them.

E.coli cells washed in cocktail of simple salt solutions achieve a state of competence during which DNA molecules may be admitted to the cell. Bacteria treated with ice cold solutions of CaCl2 and then briefly heated to 37ºC or 42ºC could be transferred with DNA.

Transformation of different bacterial strains by plasmid DNA involves the use of complex cocktails of divalent cations in different buffers, treating cells with reducing agents, adjusting the ingredients of the cocktail to the genetic constitution of particular strains of E.coli, harvesting cells at specific stages of growth cycle, altering the temperature of growth of culture before exposure to chemicals, optimizing the extent and temperature of heat shock, freezing and thawing cells and exposing cells to organic solvents after washing in divalent cations. By all these treatments transformation frequencies ranging from10 (6) to 10(9) transformants/µg of super helical plasmid DNA can be achieved.

Requirements

Reagents

  1. E.Coli (DH5α) Glycerol stock
  2. Flasks 500 ml and 250 ml
  3. Culture vessels (30 ml)
  4. Muslin cloth
  5. Luria Broth
  6. Luria Agar
  7. MgCl2
  8. CaCl2
  9. Rectified spirit
  10. Autoclave
  11. Sterilized Petriplates (disposable)
  12. Ampicillin
  13. IPTG
  14. X-gal
  15. Parafilm
  16. Spreader (glass tubes)
  17. Polypropylene centrifuge tube (30ml)
  18. Aluminium foil
  19. Ice-box
  20. Ependorff tubes (2ml)

Preparation of Reagents

  1. IPTG (stock solution, 200mg/ml) IPTG is isopropylthio-D-galactoside. Make stock solution of IPTG by dissolving 1 g of IPTG in 4 ml of distilled H2O. Adjust the volume of the solution to 5 ml with H2O and sterilize by passing it through a 0.22-µm disposable filter. Dispense the solution into 1-ml aliquots and store them at -20°C.
  2. X-gal Solution (stock solution 50 mg/ml) X-gal is 5-bromo-4-chloro-3-indolyl- -D-galactoside. Make a 5% (w/v) stock solution by dissolving X-gal in N,N’- dimethylformamide at a concentration of 50 mg/ml solution. Use a glass or polypropylene tube. Wrap the tube containing the solution in aluminum foil to prevent damage by light and store at -20°C. It is not necessary to sterilize X-gal solutions by filtration.
  3. Ampicillin (100mg/ml) Dissolve 100mg ampicillin in 1 ml of autoclaved distilled water and store at -4°C.
  4. Calcium chloride (1M) Dissolve 2.94 g of CaCl2.2H2O in a final volume of 20 ml of distilled water. Sterilize the solution by passing it through a 0.2µm filter. Store in 1 ml aliquots at -4°C.
  5. MgCl2-CaCl2 solution (80 mM MgCl2 – 20mM CaCl2Add 0.8132 g of MgCl2.6H2O in 49 ml of distilled water and 1 ml of calcium chloride 1M solution.

Equipments

  1. Laminar Air Flow (LAF)
  2. Orbital shaker incubator
  3. Autoclave
  4. Centrifuge (cooling)

Procedure

Culturing and subculturing of E. coli (strain DH5α)
Preparation of media and sterilization of media glassware and Equipments

  1. Make cotton plugs for flasks and culture vessels.
  2. Make LB media by adding 2.0 g in 100 ml of distilled water.
  3. Add 5 ml in each culture vessel and 50 ml in culture flask, Autoclave glassware and media for 15 min. at 15 psi (Don’t autoclave for more than 15 min , it can be harmful to media).
  4. Sterilize the LAF hood with UV light on for 10 minutes (Cover the LAF with glass with black paper so that UV sterilization will be more effective). Always wipe the LAF hood with rectified spirit whenever you start working and put the burner on.

Preparation of Agar Plates

  1. Dissolve 35 gm of Lurate Agar (Himedia) in 1000 ml of distilled water (app. 30 ml each for plate).
  2. Autoclave the media for 15 min. at 15 psi pressure, then add after cooling (app 50C), Ampicillin (100 µg/ml), IPTG (100 µg/ml), and X-gal (80µg/ml). (After adding ampicillin you can store the plates at 4C temperature for a week only after that the ampicillin will degrade).
    NOTE: Add ampicillin, IPTG AND X- gal only after autoclaving and cooling agar below 50°C. Ampicillin should be always stored in powder form and made fresh when needed. Solution is not stable for many days. Perform all the work in hood (LAF) with flame on and wipe the hood every time you use.
  3. After addition of IPTG, X-Gal and ampicillin, pour the media into Petri plates (up to mark, given on plate), Keep these until media gets solidifies. Seal these with parafilm.
  4. Make sure the cover of the plate should not have any moisture content. Always store the plates in inverted position at 4°C to avoid moisture accumulation inside the plate.

Inoculation and Bacterial growth

  1. Inoculate with 2 µl of E. Coli (DH5α) glycerol stock in 5ml media Discard the micropipette tip in the culture tube itself so that bacteria present inside tip can also grow. Show flame when you open the culturing vessels to vessel mouth and cotton plug. Keep cotton plug between fingers only, don’t touch
  2. Incubate these culture tubes at 37 degree with shaking at 150 rpm, overnight. Check whether culture is turbid or not, that indicates culture has grown or not.
  3. Now subculture in 50 ml media (sterilized Luria broth already prepared) with 1 ml of overnight bacterial culture, incubate at 37°C for 2-3 hrs with vigorous shaking up to when OD600 becomes app. 0.6. (as a guide line for a efficient transformation it is essential that no of viable cells not exceed 108 cells/ml, for strain DH5-α, it is OD600 0.6.to ensure that the culture doesn’t grow to a higher density, measure the OD600 of the culture every 15-20 minutes , and start harvesting the culture when the OD600 reaches 0.55.

For Bacterial growth studies
Subculture 1 ml of overnight bacterial culture in 50 ml media (sterilized Luria broth already prepared). Incubate at 37°C with shaking and measure O.D. at 600nm after every 15 min. Plot the bacterial growth curve, by plotting O.D.600 against Time.

Typical bacterial growth curve depicts four phases.

  1. Lag phase – Transfer of bacteria from one medium to another, where there exist chemical differences between the two media, typically results in a lag in cell division. This lag in division is associated with a physiological adaptation to the new environment, by the cells, prior to their resumption of division.
  2. Log phase or exponential phase – In this phase of growth, there is an increase in the cell number.
  3. Stationary phase – Stationary phase is a steady-state equilibrium where the rate of cell growth is exactly balanced by the rate of cell death (i.e., increase in cell number due to cell divisions exactly balanced by a decrease in cell number due to death).
  4. Decline phase (Death phase) – Cell death in bacteria cultures basically means that the cells are unable to resume division following their transfer to new environments.

Growth in bacteria may be compared in terms of generation time, growth rate and growth rate constant. The generation time refers to the time taken by the population to double in number. Growth rate refers to the number of generations per hour. Growth rate constant refers to the rate of growth during the growth. Growth factor is the organic compound that the organism requires from exogenous source for growth and cannot synthesize of their own. Growth in microbes can be determined by determining cell numbers, cell activity and cell mass.

Preparation of competent cells

  1.  Transfer the bacterial cells to sterile, disposable, ice-cold 50-ml polypropylene centrifugation tube. Cool the cultures to 0°C by storing the tubes on ice for 10 minutes.
  2. Recover the cells by centrifugation at 4000 rpm for 10 minutes at 4°C.
  3. Decant the medium from the cell pellets. Stand the tubes in an inverted position upon a pad of paper towels for 1 minute to allow the last traces of media to drain away.
  4. Re-suspend each pellet by swirling or gentle vortexing in 30 ml of ice-cold MgCl2-CaCl2 (80 mM MgCl2 – 20mM CaCl2) solution.
  5. Recover the cells by centrifugation at 4000 rpm for 10 minutes at 4°C.
  6. Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the last traces of media to drain away.
  7. Resuspend the pellet by swirling or gentle vortexing in 2 ml of ice-cold 0.1 M CaCl2 for each 50 ml of original culture.At this point, either use the cells directly for transformation or dispense into aliquots and freeze at -70°C.

Transformation

  1. To transform the CaCl2-treated cells store the cells for3-4 hours at 4 degree Celsius, afterwards transfer 50 µl of each suspension of competent cells to a sterile, chilled polypropylene ependorff tube using a chilled micropipette tip.
  2. Add plasmid /ligation product (no more than 50 ng in a volume of 10 µl or less) to each tube.
  3. Mix the contents of the tubes by swirling gently. Store the tubes on ice for 30 minutes.
  4. Transfer the tubes to a rack and place in a preheated 42°C circulating water bath. Store the tubes in the rack for exactly 90 seconds. Do not shake the tubes.
  5. Rapidly transfer the tubes to an ice bath. Allow the cells to chill for 1-2 minutes.
  6. Add 200 µl of LB medium to each tube. Incubate the cultures for 60 minutes in a shaker incubator set at 37°C with shaking to allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid.
  7. Transfer the appropriate volume (up to 200 µl per 90-mm plate) of transformed competent cells onto Luria Agar medium plates previously prepared containing X-gal, Ampicillin and IPTG. Spread with glass spreader uniformly until it gets resistant to move.
  8. Invert the plates and incubate at 37°C. Transformed colonies should appear in 12-16 hours.
  9. Screen the transformed colonies (White), and pick a white colony with the help of a pipette tip and inoculate into Luria Broth (5 ml) with Ampicillin in a culture tube.
  10. Incubate overnight at 37 degree Celsius with shaking. Check whether transformed colonies grown or not (by checking turbidity).Measure absorbance to check the growth.
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