Laboratory: Bacterial Transformation Introduction of plasmid DNA into E. coli E. coli

This laboratory is The first part in a series of 3 experiments: The first part in a series of 3 experiments: –Plasmid Transformation –Plasmid Isolation –Plasmid Mapping

Transformation A process of plasmid DNA uptake A process of plasmid DNA uptake In our experiment the plasmid is: extrachromosomal In our experiment the plasmid is: extrachromosomal

Transformation experiment illustrates: Genotype determines phenotype Genotype determines phenotype

Plasmid DNA How will the phenotype of the E. coli be changed?

Plasmids have selectable markers to detect change: Color alteration of colonies Antibiotic resistance

Let’s look more closely at “our” plasmid Amp r Lac Z gene pGal

What are characteristics of the lac Z gene?

Lac Z gene Codes for beta-galactosidase Beta-galactosidase is secreted by the transformed E. coli Beta-galactosidase utilizes the substrate “X-gal” to produce a blue color

What are characteristics of amp r ? What are characteristics of amp r gene?

Amp resistance gene Beta-lactamase secreted extracellularly Beta-lactamase secreted extracellularly Beta-lactamase inactivates ampicillin Beta-lactamase inactivates ampicillin

How to transform cells. Competent bacterial cells are required Competent bacterial cells are required Introduction of plasmid DNA + bacteria Introduction of plasmid DNA + bacteria “Heat Shock” to increase uptake of DNA “Heat Shock” to increase uptake of DNA

Bacterial Tranformation Protocol Protocol

Experimental overview: Please refer to your lab manual.

Group materials Each group Each group –Plasmid DNA –Buffer –Recovery broth –3 agar plates –3 transfer pipets or use micropipettors –2 “yellow platers”

Agar plate with drops of transformed cells Cell spreader Gently spread across surface Let plate sit min. Cover Incubate 37 overnight Plating of transformed bacteria

X-GalAmp/Xgal Amp/X-Gal This is in your lab manual! SUMMARY Incubate 10 min. on ice Incubate 42 C for 90 seconds Place on ice for 1 minute Add 0.75 ml recovery broth to control and treatment tubes Incubate at 37 C min streak 10 drops of cells evenly Control Treatment

Next lab: Transformation Efficiency is Determined # of transformants/ug of DNA x volume at recovery (ml)/volume plated (ml)= # of transformants/ug of DNA x volume at recovery (ml)/volume plated (ml)= # of transformants per ug of DNA # of transformants per ug of DNA Our experiment uses: DNA concentration: ug Recovery Volume:.68 ml Plating Volume: 0.25 ml