1. iGEM 101: Session 5 3/26/15Jarrod Shilts 3/29/15Ophir Ospovat

2. 5a. DNA Ligation  Seal fragments of DNA together  Formation of phosphoester bonds  5’ PO4 of one strand and 3’ OH of other strand  Complementary sticky ends hold strands in position  Vector and insert

3. DNA Ligase  T4 DNA Ligase  High efficiency  ATP dependent  Works on blunt ends (but less efficient than sticky end ligation)  Temperature dependent  Higher temperature breaks H-bonds  37 o C highest activity  Room temperature good for sticky ends  Cool temperature (~16 o C) good for blunt ends

4. Ligation Reaction  Buffer  ATP (degraded by freeze-thaw!)  Mg2+ salt  Tris  DTT (keeps enzyme in reduced form)  DNA  One part vector  Three parts insert  Ligase  Water

5. 5b. Transformation  Uptake of foreign DNA  Natural transformation (Bacillus) and chemical transformation (Escherichia)  Competent cells: M 2+ treated  Steps  Treat cells for competence  Induce DNA entry into cells (heat shock)  Outgrowth  Selection on plates  Liquid culture

6. Cell competence  Natural receptors on membrane  Uptake induced by stress  Restricted to certain species  Chemical treatment  Offset negative charges  Create holes in membrane ?  Competence lost if not kept cold  Variable degree of competence  Strain used  Preparation protocol  Handling of cells

7. DNA Uptake  Heat shock  Incubate on ice  Exactly 30 seconds at 42 o C  Briefly on ice again  Electroporation  Briefly expose to electric pulse  Disrupts membrane and introduces electrical gradient

8. Outgrowth  Grow cells in non-selective media  Recovery from shock  Time to synthesize antibiotic resistance genes  Mixed importance  Can improve efficiency several-fold  Less needed with ampicillin

9. Plating  Appropriate antibiotic resistance in plate media  Check how old plate is  Pre-warm plates while doing outgrowth  Plate culture  Swirl  Beads  Spreader  Absorption time  Incubate 37 o C  Make liquid cultures of single colonies