PLASMID DNA ISOLATION

AIM: To isolate plasmid DNA from transformed E. Coli

PRINCIPLE: Plasmid is a double stranded, circular extra chromosomal DNA of bacterium. It is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. Size of plasmids can range from 1 -1000 kbp.

  

Based on function plasmids can be of five types:

·         F/Fertility plasmid for conjugation.

·         R/Resistant plasmid which contains genes that provides resistance to antibiotics. It also helps bacteria in producing pilus.

·         Col plasmid which contain genes that code for bacteriocin (toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strains)

·         Degradative plasmid which help in the digestion of unusual substances like toluene.

·         Virulence plasmid which is responsible for pathogenicity.

Bacteria contain one or more plasmids in them. Bacteria have several mechanisms to maintain high copy number of plasmids Different plasmids have different copy numbers in the cell.

·         Relaxed plasmids are maintained in multiple copies per cell.

·         Stringent plasmids have low copy number.

Plasmid DNA may appear in one of the five conformations, which run at different speeds in a gel during electrophoresis. The conformations are listed below in order of electrophoretic mobility from slowest to fastest:

·      Nicked Open-Circular DNA has one strand cut.

·      Relaxed Circular DNA is fully intact with both strands uncut, but has been enzymatically relaxed. 

·      Linear DNA has free ends, either because both strands have been cut, or because the DNA was linear in vivo.

·      Super coiled (or Covalently Closed-Circular) DNA is fully intact with both strands uncut, and with a twist built in, resulting in a compact form.

·      Super coiled Denatured DNA is like super coiled DNA, but has unpaired regions that make it slightly less compact; this can result from excessive alkalinity during plasmid preparation.

  

Alkaline lysis is a method used in molecular biology to break cells open to isolate plasmid DNA or other cell components such as proteins.

Bacteria containing the plasmid of interest is first grown, and then lysed with a strong alkaline buffer consisting of a detergent sodium dodecyl sulfate (SDS) and a strong base sodium hydroxide. The detergent breaks the membrane's phospholipid bilayer and the alkali denatures proteins involved in maintaining the structure of the cell membrane. Through a series of steps involving agitation, precipitation, centrifugation, and the removal of supernatant, cellular debris is removed and the plasmid is isolated and purified.

Sodium dodecyl sulfate (SDS or NaDS) an anionic surfactant. The molecule has a tail of 12 carbon atoms, attached to a sulfate group, giving the molecule the amphiphilic properties required of a detergent.

 Proteins are contaminating agents in any type of DNA isolation so as in plasmid DNA isolation also. They can interfere with the final product and result in low yield. SDS is used to denature the proteins and facilitate the DNA purification process.

Agarose gel electrophoresis is a powerful separation method frequently used to analyze plasmid DNA.

The agarose gel consists of microscopic pores that act as a molecular sieve. Samples of DNA can be loaded into wells made in the gel during molding. When an electric field is applied, the DNA molecules are separated by the pores in the gel according to their size and shape. Since DNA has a strong negative charge at neutral pH, it will migrate towards the positive electrode in the electrophoresis apparatus. The rate at which a given DNA molecule migrates through the gel depends not only on its size and shape, but also on the type of electrophoresis buffer, the gel concentration and the applied voltage. Under the conditions that will be used for this experiment, the different forms of the same plasmid DNA molecule have the following rates of migration (in decreasing order): Super coiled > linear > Nicked Circles >dimer >trimer> etc.

REQUIREMENTS:

·         Micro centrifuge.

·         Water bath (37°C).

·         Automatic micropipettes with tips.

·         95-100% isopropanol Ice.

Buffers and Solutions

·         Alkaline lysis solution I.

·         Alkaline lysis solution II.

·         Alkaline lysis solution III.

·         Antibiotic for plasmid selection.

·         Ethanol.

·         Phenol: chloroform (1:1, v/v).

·         STE.

·         TE (pH 8.0) containing 20 μg/ml RNAse A.

Media:

·         Rich medium.

PROCEDURE :

1.      Fill a micro centrifuge tube with saturated bacterial culture growth in LB broth + antibiotic. Spin tube in micro centrifuge for one min. , & make sure tubes are balanced in micro centrifuge dump supernatant and drain tube briefly on paper towel.

2.      Repeat step one in the same tube filling the tube again with more bacterial culture the purpose of this step is to increase the starting volume of cells so that more plasmid DNA can be isolated per prep. Spin tube in micro centrifuge for one min. pour off supernatant on paper towel.

3.      Add 0.2ml of ice cold solution 1 to cell pellet and resuspend cells as much as possible using disposable transfer pipette.

·         Solution 1 contains glucose, tris and EDTA.

·         Glucose is added to increase the osmotic pressure outside the cells.

·         Tris is a buffering agent used to maintain a constant pH=8

·         EDTA protects the DNA from degrading enzymes (called DNAse); EDTA binds divalent cations that are necessary for DNA activity.

4.      Add 0.4ml solution 2, eppendorf tubes and inverts 5 times gently. Let tubes sit at room temperature for five minutes.

·         Solution 2 contains NaOH and SDS (detergent). The alkaline mixtures rupture the cell, and the detergent breaks apart the lipid membrane and solubilizes the cellular proteins. NaOH also denatures the DNA into single strands.

5.      Add 0.3ml ice cold solution 3, eppendorf tubes and invert 5 times gently. Incubate tubes on ice for 10 minutes.

·         Solution 3 contains a mixture of acetic acid and potassium acetate. The acetic acid neutralizes the pH allowing the DNA strands to renature. The potassium acetate also precipitates the SDS from solution, along with the cellular debris. The E coli chromosomal DNA, partially renature tangle at this step, is also trapped in the precipitate. The plasmid DNA remains in the solution

6.      Centrifuge tubes for 5 minutes. Transfer supernatant to fresh micro-centrifuge tubes using clean disposable transfer pipette. Try to avoid taking any white precipitate during the transfer. It is ok to leave a little supernatant behind to avoid accidentally taking the precipitate.

·         This fractionation step separates the plasmid DNA from the cellular debris and chromosomal DNA in the pellet.

7.      Fill remainder of the centrifuge tubes with isopropanol. Let tubes sit at room temperature for 2 minutes.

·         Isopropanol effectively precipitates nucleic acids, but is much less effective with proteins. A quick precipitation can therefore purify DNA from protein contaminants.

8.      Centrifuge tubes for 2 minutes. A milky pellet should be at the bottom of the tube. Pour of supernatant without dumping out the pellet. Drain tubes on paper towel.

·         This fractionation step further purifies the plasmid DNA from the contaminants.

9.      Add 1ml of ice cold 70% ethanol to eppendorf tubes and mix by inverting several times. Spin tubes for 1 minute. Pour of supernatant (be careful not to dump out the pellets) and drain tubes on paper towel.

·         Ethanol helps to remove the remaining salts and SDS from the preparation.

10.   Allow tubes to dry for approximately 5 minutes. Add 50µl TE to the tube. If needed centrifuge briefly to pool TE at the bottom of the tube. DNA is ready for use and can be stored indefinitely in the freezer.

OBSERVATION: The plasmid DNA was isolated successfully and was run on an agarose gel. After this the sample was viewed under UV.

RESULT: Plasmid DNA bands were observed under the UV.

Troubleshooting:

Smeared DNA bands were observed. It must have been due to the degradation of DNA by nucleases. Nucleases should be removed.

96 well-plates

COMPETENT CELL PREPARATION AND TRANSFORMATION

AIM: To prepare competent cells which are capable of taking up foreign plasmid.

PRINCIPLE:

Transformation process allows a bacterium to take up genes from its surrounding environment; that is transformation involves the direct uptakes of fragments of DNA by a recipient cell and the acquisition of new genetic characteristics.

There are two major parameters involved in efficiently transforming a bacterial organism. The first is the method used to induce competence for transformation. The second major parameter is the genetic constitution of the host strain of the organism being transformed. Competent cells are capable of taking up DNA from their environment and expressing DNA as functional proteins. If a bacterium is said to be competent, it has to maintain a physiological state in which it can take up the donor DNA. Competence results from alterations in the cell wall that makes it permeable to large DNA molecules. 

Transformation & Storage Solution (TSS) enables researchers to prepare competent E. coli in a single step and to transform the cells without heat-shock. TSS has been reported to be faster and easier than other methods of producing competent cells, such as the traditional CaCl₂ method or other high-competency protocols.

The CaCl₂ method initially produces highly competent E. coli cells, but cell competency decreases rapidly after storage at -70°C for several weeks. Other protocols produce highly competent cells that have a long storage life, but the procedures are time-consuming, requiring several transformation buffers or heat-shock steps. In contrast, TSS is a simple, one-step procedure.

Advantages of using TSS Single-step preparation of competent cells. Store prepared cells at -70°C with little or no loss in transformation efficiency. Transform cells without heat-shock. Transformation efficiencies of 106-108/µg DNA are typically obtained.

In the process of transformation, the competent cells are incubated with DNA in ice. Then it is placed in a waterbath at 42ºC and further plunging them in ice. This process will take up the DNA into the bacterial cell. Then it is plated in an agar plate containing appropriate antibiotic.

The presence of an antibiotic marker on the plasmid allows for rapid screening of successful transformants. Blue –white selection (Alpha complementation) can be used to determine which plasmids carry an inserted fragment of DNA and which do not. These plasmids contain an additional gene (lac Z) that encodes for a portion of the enzyme β – galactosidase. When it transformed into an appropriate host, one containing the gene for the remaining portion of β –galactosidase, the intact enzyme can be produced and these bacteria form blue colonies in the presence of the chromogenic substrate X – gal (5-bromo-4-chloro-3-indoyl-b-D-galactoside). 

These plasmids contains a number of cloning sites within the lac Z gene, and any insertion of foreign DNA into this region results in the loss of the ability to form active β –galactosidase. Therefore colonies that carry the plasmid with an insert (Transformants) will remain white and the colonies without the foreign DNA (Non-Transformants) will remain Blue.

We can also calculate the efficiency of transformation by using the concentration of DNA and number of transformed colonies. 
 

MATERIAL REQUIRED:

TSS solution (transformation and storage solution)

LB – 2.12gms

PEG – 10gms

100% DMSO – 5ml

1M MgCl₂ (pH 6.5) – 5ml

Weigh these components, dissolve it completely in 80ml of milliQ water and make up the volume to 100ml with milliQ water. Autoclave and store at 4⁰c (storage period: 1 week)

LB medium:

Weigh 2.5gms in 80 ml Milliq water in 500ml conical flask (head space is necessary for aeration). Dissolve it completely and make up the volume to 100ml with milliq water. Autoclave and store at room temperature.

Autoclaved dry 1.5ml eppendrof vials, sterile centrifuge bottles (250ml), dry ice with 100% ethanol.

PROCEDURE:

1.      A single colony (DH50 / sbl3) was inoculated into 5ml LB media and grown overnight at 37⁰C with shaking at 200rpm.

2.      1ml overnight culture was inoculated into 500ml conical flask containing 100ml of sterile LB broth.

3.      Culture was incubated at 37⁰c/200rpm till it reaches an OD of 0.3.

4.      The flask was removed from the incubator and kept on ice for 20 minutes.

5.      The culture was transferred to 250ml sterile centrifuge bottle and centrifuged at 1500rpm for 15minutes at 4⁰C

6.      The supernatant was discarded and pellet is restored.

7.      Carefully add 10ml of ice cold TSS solution and resuspend slowly.

8.      Aliquot 100µl of cells in 1.5ml sterile eppendrof vials and immediately flash freeze in either liquid nitrogen or dry ice containing ethanol(Note: make sure the cells are frozen immediately else the efficiency might go down).

9.      Store at -80⁰C IN cryobox with date of preparation and determine the transformation efficiency with any plasmid on the same day.

Preparing the competent cells:

Reagent: TSS (transformation and storage solution for chemical transformation)

         85% LB medium

         10% PEG (wt/vol, MW 8000)

          5% DMSO (vol/vol)

          50 mM MgCl₂ (pH 6.5)

Autoclave or filters sterilize. Store at 4⁰C for <2 weeks.

1.      Streak the cell stock on a LB plate (added antibiotic if cells have antibiotic resistant). Incubate the plate at 37⁰C overnight.

2.      Pick a single, well isolated colony and inoculate it into 5ml of LB broth (plus antibiotic). Incubate at 37⁰C overnight with shaking at 220rpm.

3.      Transfer 1ml of the standard overnight culture to a sterile 500ml flask containing 100ml of LB medium (do not add antibiotic at this step). Incubate the cells at 37⁰C with the shaking at 220rpm, until OD600 reach 0.5; this usually takes 2.0-2.5 hr. heck the OD frequently when it gets beyond 0.2 to avoid overgrowth.

4.      When the culture reaches an OD600 of 0.5, chill the flask on the ice for 20 min and the collect the cells by centrifugation at 1500rpm for 5min at 4⁰C.

5.      Resuspend the cells in 10ml of ice cold TSS solution. Now the competent cells are ready to be transformed.

6.      Aliquot 150µl competent cells to 1.5ml tube. If they are not immediately used, cells can be stored at 4⁰C for maximum of 6 hr. without significant loss of competency. The same competent cells can also be stored at -70⁰C for long term storage (pre-treat with liquid nitrogen or dry ice).

7.      Competent cells should give a minimum of 1x10⁶ tranformants per µg of plasmid DNA.

8.      Transformation frequency of frozen cells is 30% of that of the fresh cells, when used within two months.

Transforming the cells:

1.      Add DNA (20 µl) ice cold 150µl competent cells. Thaw -70⁰C competent cells first by hand temperature.

(You can transform smaller portions of cells (e.g. 75µl or 50µl), but you should decrease the volume of DNA and media proportionately.

2.      Incubate on ice for 30 min, with occasional mix.

3.      Heat shock at 42⁰C for 2 min.

4.      After heat shock, put on ice for 2 min.

5.      Add 0.8ml LB broth.

6.      Shake and incubate at 37⁰C for 60 min.

7.      Plate (<200µl) on the appropriate agar plates which contain antibiotic.

8.      Incubate plates at 37⁰C, overnight.

OBSERVATION:

The transformed cells were plated on medium containing ampicillin and the negative control plate didn’t show any growth since there were no transformed cells in that plate.  The test plate showed growth indicating the presence of transformed cells.

    

 

Positive control showed colonies

Negative control shows no colonies

Test shows few colonies

RESULT:

The organisms were transformed successfully with the plasmid inducing resistance towards ampicillin.

Troubleshooting:

Low efficiency of transformation was observed. It may be due to the improper handling of the cells. The cells should be thawed and used immediately. Refreezing of the cells can decrease efficiency. Non-optimized tubes were used. Usage of 17 x 100-mm polypropylene tubes is the best as the heat shock step is calibrated to the size and material of these tubes. If different tubes are used, the heat transfer to the cells may not be optimal. An excess DNA was used. No more that 1-10 µg of DNA should be used in 100 µl of cells. Electroporation must have being a better method.