Bacterial Transformation

Broad and Long Term Objective To characterize a single clone from an Emiliania huxleyi cDNA library using sequence analysis To characterize a single clone from an Emiliania huxleyi cDNA library using sequence analysis

Research Plan Preparation of Competent Cells and Bacterial Transformation Growth of Transformant and Plasmid MiniPrep Cycle Sequencing Sequence analysis

Today’s Laboratory Objectives 1. To prepare competent cells 2. To perform a plasmid transformation 3. To quantify transformation efficiency

Definitions “Competency” refers to the ability to take up foreign DNA some bacterial cells are naturally competent and have special proteins that are involved in DNA uptake and integration into the chromosome artificially induced competent is where DNA uptake is induced by chemical or physical means

Definitions Con’t “Transformation” is the uptake of free foreign DNA into the cell

Components of a Transformation System 1. Mode of Delivery 2. Selectable Marker 3. Propagation of Clonal Isolates

1. Chemical treatment 2. Electroporation 3. Microparticle Gun Nucleic Acid Delivery Methods

Chemical Treatment Cold Shock with high concentrations of calcium chloride and magnesium chloride Cold Shock with high concentrations of calcium chloride and magnesium chloride

Electroporation Electric Shock Opens Pores in Cell Wall

Microparticle Bombardment Shoots projectiles of gold or tungsten coated with DNA or RNA into cells using an inert gas Shoots projectiles of gold or tungsten coated with DNA or RNA into cells using an inert gas Generally used with Eucaryotes

Selectable Marker 1. Antibiotic Resistance Markers 2. Complementation using Essential Metabolic Enzymes

Map of Positive Control Vector

Map of Parent Vector pMAB58 of Experimental cDNA Clone

Ampicillin as a Seletable Marker When plated on media containing ampicillin, transformants harboring plasmids containing an ampicillin resistance gene will survive. Untransformed cells lyse in the presence of ampicillin.

Propagation of Clonal Isolates

Plasmid Transformation

Controls + Control= tests the competency of cells - Control= tests the efficacy of selectable marker - Control= tests the efficacy of selectable marker

Transformation Efficiency: Number of transformants/ng of DNA  Count number of colonies on plate after overnight growth  Determine the proportion of the total transformation that was plated (ie, volume plated/total transformation reaction volume)  Express in terms of the amount of DNA used in the reaction Number of transformants x (total transformation volume/volume plated) ng of DNA used in transformation

Next Week 1. Perform a Plasmid Miniprep 2. Quantitate Plastmid Isolated 3. Determine the Size of the Plasmid