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Cohen, Chang & Hsu. 1972. PNAS 69(8): 1110-4.

Non-chromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by R-factor DNA. Cohen, Chang & Hsu. 1972. PNAS 69(8): 1110-4. Background. Griffith et al. showed transformation of pneumococci. Background (cont’d).

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Cohen, Chang & Hsu. 1972. PNAS 69(8): 1110-4.

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  1. Non-chromosomal antibiotic resistance in bacteria: Genetic transformation of Escherichia coli by R-factor DNA Cohen, Chang & Hsu. 1972. PNAS 69(8): 1110-4.

  2. Background • Griffith et al. showed transformation of pneumococci

  3. Background (cont’d) • Avery, Macleod & McCarty (1944) showed DNA was responsible for the transformation of the R-strain

  4. What is transformation? • Transformation is the transfer of genetic material into or between organisms by extracellular DNA molecules • Brings about stable genetic changes in recipient • A transformantis a cell that has received transforming DNA and now exhibits a new phenotype

  5. Transformation • Natural transformation • Bacteria naturally take up extracellular pieces of DNA and express new characteristics encoded by that DNA • i.e. H. influenzae, B. subtilis • Engineered transformation • Bacteria must be made more permeable (competent) to extracellular DNA • May be achieved by electrical or chemical means • Electroporation • CaCl2-mediated transformation

  6. R-Factor DNA • Cohen, Chang & Hsu (1972) used R-factors • Encode antibiotic resistance functions on a plasmid • Plasmids are unique pieces of DNA Linear plasmid Circular plasmid

  7. Characteristics of Plasmids Characteristics Example • Extrachromosomal piece of DNA • Double-stranded DNA • Confers some evolutionary advantage • Origin of replication (ori) • Genes for mobilization (mob) • Great range in size • Most bacterial plasmids = 0.1 – 2kb ori camR mob Circular plasmid – 1 kb YFG

  8. Chill in CaCl2 at 0º for 20 mins -chill -collect cells -wash in CaCl2 What did Cohen et al. do? Normal E.coli Makes tiny holes in cell membrane

  9. Centrifuge Heat Shock @ 42º for 2 mins!! -Resuspend in CaCl2 -add DNA (plasmid) Incubate 0 C for 1hr!! Recovery incubation at 37º for various time points Plate directly onto agar + antibiotics

  10. Does recovery time increase resistance? • Incubated for varying lengths of time (recovery time) • allows time forKanRprotein to be expressed • kanRgene --> kanR mRNA --> KanR protein • Plated on Kanamycin plates • if still KanS --> bacteria will die • if KanR--> bacteria will live • Incubated overnight • allows single bacteria to grow into colonies • Calculated Transformation frequency • Calculating how many bacteria are actually KanR Bacteria are KanS Bacteria are KanR

  11. How to calculate transformation frequency? • Frequency = # of transformed colonies (KanR cells) # of viable cells • Example: • # of transformed colonies = 1 x 102 cells • # of viable cells = 1 x 108 cells • Calculation: • 1 x 102 1 x 108 = 1 x 10-6

  12. What influences Transformation Frequency? • Recovery time • Longer recovery time = higher transformation frequency • Maximum recovery time?

  13. Factors affecting Transformation Frequency • DNA concentration • How does DNA concentration affect transformation frequency?

  14. What else affects transformation?

  15. Does the marker make a difference? • Different drug resistance markers required different recovery times to be expressed Results after 60 minutes recovery time

  16. What just happened? • CaCl2 treatment can make bacteria competent to transformation with R-factor DNA • The number of transformants increases linearly with recovery time for one hour • Both closed and linear forms of DNA can transform cells • Damage to R-factor DNA eliminated transformation • Transformation is sharply reduced if CaCl2 cells are incubated for 2 minutes @ 42before addition of DNA ~Suggesting competent cells remain competent only for a brief period of time at elevated temperatures.

  17. What does it all mean? • Transformation of bacteria with specific genes allows us to study different bacterial processes • In some cases, can even make bacteria express human proteins!

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