1. DNA Transformation Lab E Coli with PCU

2. Avery–MacLeod–McCarty experiment Showed DNA is the substance that causes bacterial transformation

3. Hershey- Chase Showed DNA is the genetic material

4. Propagating DNA in a host cell  Requires a vector 1) Plasmid 2) Phage virus

5. Plasmids  Circular molecules of double stranded DNA that self replicating.  They code for cell functions but are not essential

6. Exchange of genes  Prokaryotes exchange genes for diversity (asexual)

7. 1) Transformation  DNA is released into surrounding medium  Recipient cells incorporate it into themselves from the medium

8. 2) Transduction  A phage virus attaches to bacterial cell and transfers its DNA into the bacterium

9. Similar???

10. 3) Conjugation  Plasmids travel between touching cells

11. R factors  Plasmids that contain resistance to antibiotics

12. Transformation in streptococcus pneumoniae in mice

13. Competent cells  Cells that can be transformed by DNA  Some can become competent by certain environmental conditions  EX. E-coli

14. E. Coli = Escherichia coli  Normally harmless bacteria in your gut  E. Coli can be artificially transformed by exposure to calcium chloride solution and thermally “shocked” by exposure to calcium chloride solution and thermally “shocked” To become receptive to foreign plasmid

15. In this lab we will perform transformation of E Coli  pUC8 = Ampicillin Resistant plasmid will be inserted into E coli

16. LAB  Sterile technique!!  Don’t touch anything  Lift Petri lid partially, pour/swab etc. Immediately reclose  Open instruments and use immediately  Do NOT touch working end  Close quickly

17. Incubation  Incubate plates UPSIDE DOWN

18. Antibiotic  Ampicillin – Kills bacteria  After mixing keep in freezer  Thaw 30 mins. prior

19. Needs Thermally shock to make E Coli competent to receive plasmid  ICE BATH followed by  Warm Water bath

20.  Bacteria Colonies  Lawn – all together not separate

21. 4 plates  Luria Broth - No antibiotic, no plasmid  Luria Broth +No antibiotic, With plasmid  AMP Broth -antibiotic, No plasmid  AMP Broth +antibiotic, With plasmid

22. What do you expect  Luria Broth - No antibiotic, no plasmid Normal growth = lawn of bacteria  Luria Broth + No antibiotic, With plasmid Normal growth = lawn of bacteria  AMP Broth - antibiotic, No plasmid NO growth, all killed  AMP Broth + antibiotic, With plasmid Individual Colony growth of transformed resistant bacteria

23. results

24. Calculating efficiency of transformation Total mass of plasmid used Total mass = volume X concentration Volume = 10 µl Concentration = 1 µg/100µl = 0.1 µg plasmid

25. Total volume of suspension  Volume calcium chloride (chilled)  Volume Plasmid  Volume Luria broth  TOTAL Volume of suspension .25 ml = 250µl calcium chloride  10 µl Plasmid .25 ml = 250 µl luria broth  Total = 510 µl

26. Fraction of suspension put on plate  µl on plate/total volume .1 ml put on plate =100 µl  Total volume = 510 µl (from above)  100 µl x 510 µl = 0.196 µl put on plate

27. Total mass of plasmid in fraction  (mass of plasmid x fraction on plate)  Mass of plasmid = 0.1 µg  Fraction on plate = 0.196 .1µg x 0.196 =  Total mass of plasmid on plate = 0.0196 µg

28. Number of colonies per µg of plasmid  (# colonies counted/mass of plasmid put on plate)  Count = #  Mass of plasmid = 0.0196 µg  Colonies per µg plasmid = …………