Lecture 4 – Methods of Gene Transfer PDF

Lecture 4 – Methods of gene transfer PREPARED BY: DR MOT YEE YIK LECTURER FACULTY OF APPLIED SCIENCES Lecture contents  Methods of recombinant gene delivery  Bacterialtransformation  Electroporation  Types of selection markers  Antibiotic resistances genes  LacZ ge...

Lecture 4 – Methods of gene transfer PREPARED BY: DR MOT YEE YIK LECTURER FACULTY OF APPLIED SCIENCES Lecture contents  Methods of recombinant gene delivery  Bacterialtransformation  Electroporation  Types of selection markers  Antibiotic resistances genes  LacZ gene Learning outcomes At the end of this lecture, students will be able to:  Describe the methods of gene transfer into host cells  Describe the different selection markers used for identifying positive clones How do we get vectors into host cells?  Bacterial transformation The common methods of introducing free DNA into a host cell include: 1. CaCl2 - heat transformation Bacteria cells are treated with ice cold CaCl2 and then exposed to high temperature (42-45°C) for about 45-90 seconds. 2. Electroporation electric field may be used to render the host cell wall permeable to the free DNA.  The concept of CaCl2 or heat transformation 1. E. Coli bacterial cells are treated with CaCl2 (which helps plasmid attach to bacteria) CaCl2 treated Plasmid E. coli e.g.pBlu 2. Plasmid are co-incubated together with bacteria in the same microtube for DNA transfer Procedure of Recombinant DNA Bacterial Transformation Recombinant Transformation 1 1. Prepare competent cells 2. Competent cells are kept frozen at -80°C 3. When required, competent cells are thawed on ice and flicked to resuspend Recombinant Transformation 2 1. Open the tube of competent cells and add 4 µl of the ligation solution, directly to the tube solution using a micropipettor and sterile tip. 2. Close the tube. Note: Ligation solution consists of RE digested plasmid and recombinant DNA which has been ligated with ligase enzyme Recombinant Transformation 3  Place the tube on ice for 30 minutes. Why do we do this?  Recombinant Transformation 4  Remove the tube from the ice and immediately hold it in a 42°C water bath for 45 seconds.  Place the tube directly back on ice for 1 minute. Why do we do this? Recombinant Transformation 5  Use a sterile pipette to add 10 drops of sterile LB nutrient broth to the competent cell tube.  Mix by tipping the tube and inverting it gently. Why do we do this?  Recombinant Transformation 6  Incubate the mixture for 3-4 hours in a water bath or incubate on 37°C shaker for 45 minutes. Why do we do this? Recombinant Transformation 7  Label your LB agar plates with your name and date  The agar should have the appropriate antibiotics in them (the plasmids transformed into the competent cells should be resistant to this antibiotic) Recombinant Transformation 8 1. After the incubation period, we pellet down the cells by short spin at 3K rpm. 2. Resuspend the pellet with around 200 μl of supernatant. 3. Place the cell suspension onto the center of each of the agar plate. 4. Use a sterile cell spreader to spread the liquid evenly across the surface of the agar plate. 5. Be careful not to touch the part of the cell spreader that comes in contact with the agar (ensure sterility).  Recombinant Transformation 9  Incubate the plates (agar down) for 2 hours and then invert (agar up) for the next 24-36 hours in a 37°C incubator. Why do we do this? How will we know if the plasmid has been transformed into the bacteria? Transformation produces bacteria with: 1. Religated plasmid 2. Religated insert Which one will 3. Recombinant plasmids grow in your antibiotic agar plate? Why use selectable markers?  Is required due to inefficiency of ligation and bacterial transformation.  Even with high efficiency systems - 1/1000 of the bacterial cells take up the DNA.  Importance of markers:  To prevent the non-transformed (non competent) cells from growing.  To later Differentiate transformed cells containing plasmids with DNA inserts from plasmids with no DNA inserts (with additional tests) Antibiotic resistant genes 1 1. The Antibiotic resistance gene permits ascertaining successful transformation. Plating out the transformation mix on an agar plate containing the relevant antibiotic, and only the transformants will be able to grow. 2. A second antibiotic-resistant gene can also be included to ascertain insertion of foreign DNA. Antibiotic resistant genes 2 Using the tetracycline and ampicillin resistant plasmid 1. Grow cells with tetracycline so only cells with plasmid grow (unaffected resistant gene) 2. Next, grow copies of the original colonies with ampicillin which kills cells with plasmid that contains the foreign DNA (the affected gene)  Antibiotic resistant genes 3  Screening With Replica Plating  Replica plating transfers clone copies from original tetracycline plate to a plate containing ampicillin  A sterile velvet transfer tool can be used to transfer copies of the original colonies  Desired colonies are those that do NOT grow on the new ampicillin plate  Selectable markers : 2. Lac Z gene  Use agar medium containing an antibiotic and the chemical X-gal.  The Lac Z gene in the plasmid vector produces beta- galactosidase which metabolized X-gal to produces a blue product, Indo Blu.  Only bacterial that contains the plasmid with the antibiotic- resistance gene can grow.  Of these colonies, those with an intact Lac Z gene (and therefore no insert) will produce blue colonies.  Those with a disrupted LacZ gene (and therefore with an insert) are unable to metabolize X-gal and produce white colonies.  Therefore the white colonies contain recombinant plasmids  pBlu plasmid containing the Lac Z gene RNA This plasmid contains genes for: Protein that allows for 1. AMP = ampicillin (an antibiotic resistance antibiotic) resistance 2. Beta galactosidase an enzyme that converts X-Gal Indo Blu Enzyme that breaks RNA down X-Gal to make Indo Blu Thank you for your attention