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Wet Lab: EcoRV Restriction Endonuclease over expression and digestion of DNA
Restriction endonucleases are found throughout the prokaryotic world and function as primitive immune systems to degrade the DNA of invading viruses and bacteriophage. They consist of a restriction enzyme and a DNA methyltransferases enzyme (Figure 1).
Restriction enzymes typically identify a specific DNA sequence, bind to it, and using Mg2+ as a cofactor catalyse the cleavage of both strands of the DNA. They are usually dimeric enzymes, with one half of the enzyme recognising one half of the palindromic recognition sequence: the symmetry of the enzyme is reflected in the symmetry of the substrate.
File:ImperialCollege Spring2008 SyntheticBiology EcoRIV.jpg
To avoid digestion of the host DNA, alongside the restriction endonuclease gene is the gene for a DNA methylase. The DNA methyltransferases enzyme binds to the same sequence of DNA as the restriction enzyme, but it catalyzes the transfer of a methyl (CH3) group from its cofactor, S-adenosylmethionine, to either an adenine (A) or cytosine (C) within the recognition sequence. Since, following DNA replication one of the strands will already be methylated, the DNA methyltransferases will only ever have to methylate one strand of a hemi-methylated DNA in the host genome. For this reason the enzymes function as monomeric proteins.
Once both strands of the recognition sequence are methylated the site is refractory to cleavage by the restriction endonuclease. Should a bacteriophage attempt to invade the cell, then its DNA will be un-methylated and liable to cleavage by the host restriction enzyme, which are maintained at higher levels than the methyltransferases.
Restriction endonucleases have proved to be an essential discovery for recombinant DNA technology, and was recognised by a Nobel prize for Werner Arber and Ham Smith. They are extremely specific enzymes, which means they can be used to cut DNA very precisely at known positions. With the use of DNA ligase we can therefore cut DNA up in very precise ways and rejoin it to make new DNA constructs.
In this project you will be provided with a strain of E. coli that contains two plasmids, one contains the EcoRVm gene, which is constitutively expressed. The other contains the EcoRVr gene, which is inducible from the Pr promoter. When this plasmid is introduced into non-lysogenic strains (which do not have the phage integrated into the host genome), the restriction enzyme is expressed since it will not be expressing the repressor. However, over expression of the endonuclease is lethal to E. coli, even in the presence of the methylase, since in very high concentrations it will begin to cleave at other (non-cognate) DNA sites. For this reason, the strain is grown at 28°C up to mid-log phase and then expression is increased by raising the temperature to 42°C for 4h. During this time the cells are able to express a significant quantity of the enzyme before they become too damaged.
You will grow an overnight culture by inoculating a liquid culture from a single bacterial colony taken from a plate. This colony has grown from an individual E. coli cell, and this ensures that you will be growing a culture isolated from a genetically homogenous sample. The overnight culture will then be used to inoculate a larger culture. This will then be induced by increasing the temperature. The cells will be harvested at the end of the day and frozen. You will then lyse the cells to release the restriction enzyme, make serial dilutions of the enzyme and use these to digest a sample of DNA, which will then be run on an agarose gel to visualise the restriction fragments.
- 2 x 30 ml sterilin universals
- 2 Inoculating loops
- 1 ml cuvettes for cell density measurements
- 4 x 250 ml flasks with 25 ml LB media
- 4 x foam bungs
- Aluminium foil
- 2 x 50 ml centrifuge tubes
- Dry ice
- 1.5 ml eppendorfs
- P20 Gilson
- P200 Gilson
- P1000 Gilson
- Overnight autoclave (mon)
- Shaking incubator at 28°C (tues)
- Water bath at 55°C (for 30 250 ml flasks) (tues)
- Spectrophotometer for OD550 measurements (tues)
- 25 ml pipette (tues)
- centrifuge for 50 ml tubes (tues)
- 25°C waterbath for eppendorfs (Thurs)
- 37°C waterbath for eppendorfs (Thurs)
- Microcentrifuge (thurs)
- Agarose electrophoresis tanks and powerpacks (thurs)
- 1000 x Ampicillin stock solution (50 mg/ml)
- 10 ml LB medium autoclaved
- 1M Tris-HCl pH 7.5
- 5 M NaCl
- 0.5 mM EDTA pH 8.0
- 1 M MgCl2
- 10 mg/ml BSA (bovine serum albumin)
- Lysozyme 100 mg/ml in 50 mM Tris pH 8.0 (20 l per student)
- DNA (500 g/ml)
- 1xTAE (or 0.5 x TBE)
- Sucrose loading dye with bromophenol blue