lec 7-2024.ppt

Introductory Molecular Biology Microbiology 202 – July 2024 Prof Boshoff, Biotechnology Innovation Centre Lecture 7 Outline of schedule 17 July Determination of the nature of the genetic material in bacterial cells: Enzymatic diges...

Introductory Molecular Biology Microbiology 202 – July 2024 Prof Boshoff, Biotechnology Innovation Centre Lecture 7 Outline of schedule 17 July Determination of the nature of the genetic material in bacterial cells: Enzymatic digestion of cell extracts and gel electrophoresis prac starts at 12.10 today! 24 July Transformation of Escherichia coli competent cells 25 July Count the colonies on the plates after overnight incubation.  INTRODUCTION The aim of these experiments is to give you an opportunity to retrace the steps of early microbiologists who set out to determine the nature of the genetic material. You will be provided with a cell-free extract from E. coli cells resistant to the antibiotic ampicillin consisting mostly of DNA, RNA and protein. Preparation of cell extract for transformation Aliquot 10 μl of the cell extract into each of four, sterile eppendorf tubes (labeled 1-4). Add 10 μl sterile TE buffer to tube no 1 (control). Add 10 μl DNase mix to tube no 2. Using a fresh, sterile pipette tip, add 10 μl RNase to tube no 3. Use a fresh, sterile pipette tip; add 10μl protease mix to tube no 4. Vortex the tubes briefly to mix the solutions and incubate tubes 1 - 4 at 37oC in a water bath for 120 minutes. (over lunch) Remove from the water bath and put the tubes on ice. Centrifuge briefly (30 sec) Gel electrophoresis and visualization of nucleic acids Pipette four droplets (10 μl each) of gel tracking dye onto the parafilm provided. Do this next to where your gel apparatus has been set up. Using a fresh, sterile pipette tip each time, carefully remove 10 μl from tube 1 and mix with a droplet of tracking dye on the parafilm surface by drawing the liquid up and down the pipette tip a few times. Do the same with 10 μl of tube 2, 3 and 4. You should now have four samples of extract mixed with dye. Freeze the remaining samples in tubes 1 - 4 at -20oC for use during part 2 – label carefully and place in freezer box Gel Electrophoresis separates macromolecules - size, electric charge and other physical properties electrophoresis = migration of charged particles under the influence of an electric field Agarose gel electrophoresis - physical method for determining the size of DNA -agarose - linear polysaccharide extracted from seaweed - inter- and intramolecular hydrogen bonding within and between agarose chains - separation based on resistance caused by gel matrix gel material acts as a "molecular sieve", separating the molecules by size. Agarose gel electrophoresis Gels – porous, size of the pores relative to the molecule determines if it enters the pore and is retarded or will bypass it. separation - charge on the molecule and size. When current is applied - molecules separated accordingly to molecular size and move to their respective electrodes, tracked by dye. nucleic acids run to anode (+) why? - 0.3 to 2% agarose - the higher the agarose concentration the "stiffer" the gel, creates a denser sieve to increase the separation of small DNA length differences -DNA of 200 to 50,000 bp can be separated -Different forms of DNA move through the gel at different rates -DNA - more compact shape moves faster compared to linear DNA of the same size - mix agarose with buffer solution, melt it by heating, and pour the gel - Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE) - buffers establish a pH and provide ions to support conductivity - loading buffer – contains glycerol and a tracking dye (bromophenol blue) Visualization Methods - nucleic acids cannot be visualized directly -ethidium bromide and Gel Red intercalate between stacked nucleic acid bases - UV light causes flourescence in orange-red light range - bromophenol blue - track the progress of the electrophoresis Preparing the Gel Add agarose to 0.5x TBE buffer in an Erlenmeyer flask Heat the slurry in a microwave oven until the agarose dissolves Cool the mixture to 50 - 60°C, add ethidium bromide and mix thoroughly Preparing the Gel Pour the solution into the gel-tray and allow the gel to set. Remove the comb and place the gel in the electrophoresis tank. Add enough 0.5x TBE buffer to cover the gel  The migration rate of linear fragments of DNA is inversely proportional to log 10 of their size in base pairs, the smaller the linear fragment, the faster it migrates through the gel. The exact sizes of separated DNA fragments can be determined by plotting the log of the molecular weight for the different bands of a DNA standard (DNA ladder) against the distance travelled by each band. Carefully load the samples into the wells of the agarose gel. The blue mixes will sink to the bottom of the well. With the help of your tutor, connect the electrodes and switch on the current. Allow the gels to run for approximately 1 hour at 100 V. Under supervision of your tutor, remove the gel and visualize the DNA on a UV transilluminator. Photograph the gel using the departmental gel documentation system. The tutor will demonstrate the use of the UV illuminator and gel documentation system. The DNA and RNA will be stained with ethidium bromide, which fluoresces when it is exposed to ultraviolet light. Thus the DNA and RNA in your sample will be visible as bright bands in an otherwise unstained background. Your tutors will photograph your gel for you - give your tutor your email address Present this in your notebooks with the following annotations: Lane numbers, Arrows showing the position of the DNA and RNA on the gel, Figure legend containing detailed description of the contents of each lane. Gel electrophoresis and visualization of nucleic acids Done – get pictures of your gels Freeze the remaining samples in tubes 1 - 4 at -20oC for use during part 2 Transformation of competent E. coli cells Next week DNA replication DNA is a template for its own replication Three models for DNA replication: Conservative - parent chromosomes remain intact Semi-conservative - daughter chromosomes consist of one parental strand and one nascent strand Dispersive - parental strands dispersed between the daughter strands We know that DNA replication is semiconservative, due to time constraints we won’t look at the experiment that proved this hypothesis (Meselson–Stahl Experiment, 1958) The Meselson–Stahl Experiment may be covered during Biochemistry 3 lectures DNA is a template for its own replication. Each parallel DNA strand acts as a template for DNA replication to form a complementary daughter strand. The two resulting double helices contain one parental strand and one new (nascent) strand. Replication of the E. coli chromosome and plasmids (prokaryotes) - chromosome is ds, circular - semi-conservative replication begins at a fixed point origin of replication (oriC) – bind initiator proteins DnaA - replication is bidirectional - oriC and termination sites (ter) are on opposite sides - can be replicated in about 40 min Replication of eukaryotes - chromosome is ds, linear - replication begins at many places, more than one ori up to 20000 in mammalian cells - replication is bidirectional - replication at extreme ends is difficult - telomeres Telomeres caps at the end of chromosome, like the plastic tips at the end of shoelaces Telomeres shorten as we age (also stress, no exercise, obesity) Telomerase - germline and stem cells, maintains telomere length by adding 'TTAGGG' repeats to the ends of chromosomes. The enzymes used for replication DNA polymerases catalyse DNA synthesis in 5’ to 3’ direction It requires – a template, a primer to provide free 3’ OH, dNTPS E. coli also has 5 different polymerases, more than 15 in eukaryotes DNA polymerase III plays a major role in DNA replication, DNA polymerase I also involved to a lesser extent DNA polymerase III DNA polymerase III consists of ten subunits all of which are required for function of the holoenzyme, Asymmetric dimer 2 core enzymes (αεθ) α (alpha) – polymerase activity (encoded by dnaE gene) ε (epsilon) - exonucleolytic proofreader 3'→5' exonuclease activity (dnaQ) θ (theta) - stabilizer for ε and stimulates 3'-to-5' exonuclease activity Each core binds one strand of DNA Associated with each core is β clamp (dnaN) (donut-shaped ring) – tethers core enzyme to DNA, helps enzyme to replicate DNA without “falling off” At the centre is the clamp loader, used to load β clamp onto DNA, uses ATP when loading beta onto primed DNA A dimer of τ (tau) holds holoenzyme together, activates DnaB helicase activity  Figure 5-31 Action of DNA polymerases. Page 99 nucleophilic attack by OH on α-phosphate oriC