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 DNA Sequencing Sequence analysis

Today’s Laboratory Objectives 1. To prepare competent bacteria bacteria 2. To perform a transformation of competent bacteria with competent bacteria with plasmid DNA plasmid DNA 3. To quantify transformation efficiency efficiency

Definitions “Competency” refers to the ability to take up DNA Some bacterial cells are naturally competent and readily take up DNA from the environment Bacterial competence can be artificially increased by by chemical or physical means “Transformation” is the uptake of free foreign DNA into the cell foreign DNA into the cell

Transformation of chemically competent bacteria Incubation with high concentrations of cold calcium chloride followed by a brief heat shock Incubation with high concentrations of cold calcium chloride followed by a brief heat shock

Other DNA delivery methods: Electroporation Electric shock opens transient pores in cell membrane

Shoots projectiles of gold or tungsten coated with DNA or RNA into cells Shoots projectiles of gold or tungsten coated with DNA or RNA into cells Generally used with eukaryotic cells Other DNA delivery methods: Microprojectile bombardment

Plasmids 1. Origin of replication - site of initiation of plasmid replication - determines copy number 2. Selectable marker genes - antibiotic resistance markers - complementation using essential metabolic enzymes metabolic enzymes 3. Gene of interest

Map of Parent Vector pMAB58

Ampicillin as a Selectable 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

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 colony forming units/μg of added DNA - - Compare your transformation efficiency with the listed transformation efficiency of a commercial preparation of competent cells (e.g. Invitrogen, Promega)