Answer to Question #118315 in Biochemistry for kwesi

Question

b. Elucidate two physical properties that will enable you to isolate TOL plasmid from the chromosomal DNA of the bacteria

Answer 1

Originally evolved from bacteria, plasmids are extrachromosomal genetic elements present in species of Archae, Eubacteria and Eukarya which can replicate independently. Plasmids are circular double stranded DNA molecule that are distinct from cell's chromosomal DNA.

Structure and function of a bacterial cell is directed by genetic material conatined within chromosomal DNA. Plasmids contribute to bacterial genetic diversity and plasticity by encoding functions which may not be specified by bacterial chromosomal DNA. As a tool, plasmids can be modified to express the protein of interest.

Plasmids have served as invaluable model systems for the study of processes like DNA replication, segregation, conjugation and evolution. Plasmids present in the bacterium differ in their physical properties like in size,

geometry and copy number.

Although most plasmids possess a circular geometry, there are now examples of plasmids tht are linear in a variety of bacteria. Plasmid DNA may appear in one of the five conformations 'nicked open circular DNA' which has one strand cut, 'relaxed circular DNA' is fully intact with both strands uncut but has been enzymatically relaxed, 'linear DNA' has free ends, 'supercoiled DNA' is fully intact with both strands uncut and 'supercoiled denatured DNA' is like 'super coiled DNA' but has unpaired regions which make it less compact.

Plasmid Copy Numbers:

Copy number refers to average or expected number of copies per host cell. Plasmids are either low, medium or high copy number. Knowing which category the plasmid falls under is significant when starting out an experiment. If working with a low-copy number plasmid which is associated with a low yield and may thus be required to set up more cultures. If a poor yield is obtained from a high copy plasmid, troubleshooting is required. In bacterium with high copy number plasmids, during cell division plasmids get segregated randomly in daughter cells whereas in case of bacterium with low copy numbers, during cell division the plasmids divide equally in daughter cells. An advantage of high copy number is greater stability of plasmid when random partitioning occurs at cell division.

Plasmid Isolation:

The isolation of plasmid DNA from bacteria is a crucial technique in molecular biology and is a necessary step in procedures like cloning, DNA sequencing, transfection and gene therapy. These manipulations require the isolation of high purity plasmid DNA. Purified plasmid DNA can be used for immediate use in all molecular biology processes.

Alkaline lysis is a method used in molecular biology, to isolate plasmid DNA or other cell components like proteins by breaking the cells open. Bacteria containing the plasmid of interest is first is first grown and then allowed to lyse with an alkaline lysis buffer consisting of a detergent sodium dodecyl sulphate (SDS) and a strong base sodium hydroxide. The detergent cleaves the phospholipid bilayer of membrane and the alkali denatures the proteins which are involved in maintaining the structure of cell membrane. Through a series of steps involving agitation, precipitation, centrifugation and removal of supernatant, cellular debris is removed, the plasmid is isolated and purified.

Principle:

Purification of plasmid DNA from bacterial DNA is based on the differential denaturation of chromosomal and plasmid DNA using alkaline lysis in order to separate the two. Basic steps of plasmid isolation are disruption of cellular structure to create a lysate, separation of the plasmid from chromosomal DNA, cell debris and other insoluble material. Bacteria are lysed with a lysis buffer solution containing sodium dodecyl sulphate and sodium hydroxide. During this step, disruption of most cells is done. Chromosomal and plasmid DNA are denatured and resulting lysate is cleared by centrifugation, filtration or magnetic clearing. Subsequent neutralization with potassium acetate allows only the covalently closed plasmid DNA to reanneal and to stay solubilized. Most of the chromosomal DNA and proteins precipitate in a complex formed with potassium (K) and SDS, which is removed by centrifugation. Bacteria is suspended again in a re-suspension buffer (50mM Tris-Cl, 10 mm EDTA, 100 µg/ml RNase A, pH= 8.0) and treated with 1% SDS (w/v) or alkaline lysis buffer (200 mM NaOH) to liberate plasmid DNA from E.coli host cells. Neutralization buffer solution (3 M potassium acetate, pH 5.0) neutralizes the resulting lysate and creates appropriate conditions for binding of plasmid DNA to silica membrane column. Contamination like salts, metabolites and soluble macromolecular cellular components are removed by simple washing with ethanol wash buffer (1.0 M NaCl, 50 mM MOPS, pH 7.0, isopropanol 15%). Pure plasmid DNA is lastly eluted under low ionic strength conditions with alkaline buffer (5 mM Tris/HCl, pH 8.5).

Culture Media:    

Yield and quality of plasmid DNA depend on the type of culture media used. Most of the plasmid purifications are optimized with cultures grown in standard Luria Bertani (LB) medium. For LB medium preparation, one has to dissolve 10g tryptone, 5 g yeast extract and 10 g NaCl in 800 ml distilled water. Adjust the pH to 7.0 with 1N NaOH. Adjust volume to 1 liter by adding distilled water and sterilize by autoclaving. Cell culture should be incubated at 37°C with constant shaking for 12-16 hours overnight. An OD of 3-6 can be achieved.

Care needs to be taken as overgrowing a culture may lead to a higher percentage of dead or starving cells and resulting plasmid DNA may be partially degraded or contaminated with chromosomal DNA. To find optimal culture conditions, culture medium and incubation times have to be optimized for each host strain/plasmid construct combination individually.

Lysate and Neutralization:

Lysis formulas may vary depending on whether one wants to extract DNA/RNA/plasmid. All the methods of lysing bacteria will yield plasmid solutions contaminated with chromosomal DNA and RNA. Centrifugation removes vast majority of chromosomal DNA (it will form a pellet while plasmid DNA remains soluble) and treatment with RNase will eliminate RNA contaminant.

Lysis buffers contain a high concentration of chaotropic salts. Chaotropes have two important roles in nucleic acid extraction. Firstly, they destabilize hydrogen bonds, Van der Waals forces and hydrophobic interactions, leading to destabilization of proteins, including nucleases. Secondly, they disrupt association of nucleic acids with water, thereby providing optimal conditions for their transfer to silica. Separation & removal of plasmids from bacterial cell is brought about by re-suspension of 1-5 mL of culture in a re-suspension buffer solution (50mm Tris-Cl, 10 mm EDTA, 100 µg/ml RNase A, pH= 8.0) and pellet cells in a micro centrifuge at 11000x g for 30 seconds.

Lysate is achieved by adding 250 µL of lysis buffer with neutralization buffer, as it aids in complete precipitation of SDS, protein and genomic DNA. Incomplete neutralization leads to reduced yield. However, released plasmid DNA is very vulnerable at this stage and shaking too much will damage the DNA.

Binding and Washing in silica membrane:

After centrifuging the lysate through silica membrane, desired nucleic acids must be bound to the column and impurities like protein and polysaccharides should be in the flow-through. Plant samples will contain harides and pigments, while for blood samples, the membrane may be slightly brown or yellow in colour. The wash steps will remove these impurities. There are two wash steps. First wash will include a low concentration of chaotropic salts to remove residual proteins and pigments. This is followed with an ethanol wash to remove salts.

Columns contain a silica resin which selectively binds to DNA/RNA. The DNA of interest can be isolated by virtue of its ability to bind silica in presence of high concentrations of chaotropic salts. These salts are then removed with an alcohol based wash and the DNA is eluted using a low-ionic-strength solution like TE buffer or water. Binding of DNA to silica seems to be driven by dehydration and hydrogen bond formation, which competes against weak electrostatic repulsion. Thus, a high concentration of salt will help drive DNA adsorption onto silica and a low concentration will release the DNA.

 Elution:

The elution buffer volume and method can be adapted to subsequent downstream application to achieve higher yield and/or concentration than the standard method. Elution buffer is used to wash away unbound proteins at first and at a greater concentration, it releases the desired protein from the ligand. The elution buffer should work quickly without changing the function or activity of desired protein. For maximal DNA elution, allow the buffer to stand in the membrane for a few minutes before centrifugation.  Elution buffer (5mM Tris/HCl, pH 8.5) can be replaced by TE buffer. Using a weakly buffered alkaline buffer containing no EDTA is preferred particularly if the plasmid DNA is intended for sequencing reactions.