ISOLATION OF CHROMOSOMAL DNA FROM BACTERIA

Introduction:
The bacterial cell does not have its genetic material enclosed in a membrane bounded nucleus and hence is a prokaryote. Instead, they contain highly compact structure called nucleoid.The chromosome of E. coli and presumably of most bacteria is a single super coiled double-stranded circular DNA molecule complexed with a specific amount of protein and variable amount of RNA.The E.coli chromosome is well studied. For E. coli the total length of the circular chromosomal DNA is about 1300 m where as the rod-shaped bacterium has a diameter and length of about 1 and 3 m, respectively. Therefore the bacterial chromosomal DNA must be highly folded when it is in a cell. The genome content is about 5000 kbp consisting of 2800 genes. Two important features of the structure of E. coli chromosome have been revealed by electron micrograph studies, a) The DNA is arranged in a series of loops and b) each loop is super coiled. The physical organization of bacteria (prokaryotes) is simple as compared to eukaryotes. In bacteria, there are two distinct classes of cell envelopes known as Gram-positive and Gramnegative. Gram-positive cell envelopes consist of plasma membrane and peptidoglycan layer (cell wall) where as the architecture of Gram-negative cell envelope is more complex consisting of plasma membrane, periplasmic space, peptidoglycan and outer membrane.E.coli is an example of Gram-negative bacterium. Therefore its cell wall is more complicated than that of Gram-positive cells. In order to isolate chromosomal DNA from E. coli, the cell envelope structure needs to be weakened and ruptured. Treatment of cell with various reagents such as trypsin (which hydrolyze proteins), detergents (which remove lipids), and lysozyme (muramidase) causes dissolution of peptidoglycan layer. The isolation of E.coli chromosomal DNA is based on the following principle:

Principle:
The cells are first digested with lysozyme in order to weaken the cell walls then ruptured with the detergent sodium doddery sulphate. In the extraction medium high salt concentration (0.15 mol/litre helix NaCl) is used. This helps to prevent strand separation of the double of the DNA.EDTA is also present and his chelate metal ions (such as Mg++, Ca++) needed for DNase and so inhibits the activity of the enzyme. Protein is denatured by the treatment with the buffersaturated phenol. Extraction is carried out with the organic solvent mixture (phenol: chloroform: isoamyl alcohol) to remove denatured protein contaminants isoamyl alcohol is present as an antifoaming agent. After removal of the protein, DNA is removed by the precipitation with ethanol or isopropanol.Further incubation of the chromosomal DNA solution with RNase (this should be DNase free by heat activation) should ensure that contamination of the DNA by RNA is kept at minimum level. The conc. Of the isolated DNA may be determined by UV absorption spectrum at 260 nm and 280 nm can be used to detect protein contamination. The quality of the chromosomal DNA may be further assessed through digestion by restriction enzymes followed by agarose gel electrophoresis.

Requirements:
(Media and solutions should be sterile. Sterilization by autoclaving needs to be done at 15 lbs for 15 minutes) E. coli DH5 strain,LB medium and LB plates, Saline EDTA (0.15M NaCl,0.1M EDTA adjusted to pH 8.0), Lysozyme soln.(10 mg/ml),1.0 M Tris.HCl (pH 8.0),10% sodium dodecyl sulphate (SDS),50 mM Tris buffer (pH 8.0),saturated phenol,Chloroform,isoamyl alcohol,TE buffer (10 mM Tris HCl pH 8.0,1 mM EDTA pH 8.0),Iso propanol, Dehydrated ethanol ,DNase free RNase solution(10 mg/ml),microfuge tubes, micro tips, UV spectrophotometer and quartz cuvettes

Procedure:
The following protocol may be used for small-scale preparation of E. coli chromosomal DNA: Pick a single colony of E. coli DH5 strain from a freshly grown plate and transfer it into 20 ml of LB broth in a 250 ml of flask. Incubate the culture for 16-20 hours at 37C with vigorous shaking (200-250 cycles/minute in a rotary shaker).(More than one group may be involved in the same isolation procedure with this culture). Harvest cells from the above 1.5-2.0 ml stationary phase culture in sterile microfuge tube. Decant the media from the cell pellets, stand the tubes in an inverted position for one minute to allow the last traces of media to drain away(The cell pellet may be washed here using 10mM Tris HCl pH 8.0) Resuspend each cell pellet in approximately 0.8ml saline EDTA buffer thoroughly. Add 50 l freshly prepared lysozyme soln.mix well. Incubate at 37C for 20 minutes. Add 0.2 ml 10% SDS, mix well by inversion, and incubate in water bath at 60C for 15 minutes. Extract once with buffer-saturated phenol. Extract once with phenol: chloroform: isoamyl alcohol (25:24:1) Transfer the upper aqueous phase to a sterile microfuge tube. Precipitate DNA by adding equal volume of isopropanol. Wind out the DNA fibers on the glass rod. Squeeze as much liquid as possible from the spooled mass by pressing it against the side of microfuge tube. Dissolve the DNA fibers in 500 l TE buffer Add DNase free RNase solution to a final conc. of 40 g/ml and incubate at 37C for 30 minutes, with occasional shaking. Extract once with equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) Precipitate DNA from the upper aqueous layer with 2 volume of ethanol. Spool out the DNA and redissolve it in 50 l TE (pH 8.0).Store at 4 C for further use.

Calculate the conc. of DNA using UV spectrophotometer (50 g/ml of DNA) has an extinction of 1.0 at 260 nm in 1 cm cuvette).Also find out the E260/E280 ratio.

References:
Oishi, M and Cosloy, SD.1972.The genetic and biochemical basis of the transformability of Escherichia coli K12.Biochem.Biophys.Res.Cummun.49:1568 Worcel, A.and Burgi, E.1972.On the structure of the folded chromosome of Escherichia coli.J.Mol.Bio.71:127 Sinden, RR.And Pettijohn, DE.1981.Chromosomes in living Escherichia coli cells are segregated into domains of supercoiling.Proc.Natl.Acad.Sci. USA 78:224 Plummer, DT.1988.an introduction to Practical Biochemistry (Chapter 16), (Third Edition),Tata McGraw-Hill, New Delhi.