DNA Transformation Lab E Coli with PCU

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

Hershey- Chase Showed DNA is the genetic material

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

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

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

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

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

Similar???

3) Conjugation  Plasmids travel between touching cells

R factors  Plasmids that contain resistance to antibiotics

Transformation in streptococcus pneumoniae in mice

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

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

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

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

Incubation  Incubate plates UPSIDE DOWN

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

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

 Bacteria Colonies  Lawn – all together not separate

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

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

results

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

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

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 = µl put on plate

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

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