Bacterial Transformation

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Bacterial Transformation
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Broad and Long Term Objective

• To characterize a single clone from an
  Emiliania huxleyi cDNA library using sequence
  analysis

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Research Plan
Preparation of competent cells and bacterial
transformation
Growth of transformant and Plasmid MiniPrep
DNA Sequencing
Sequence analysis
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Todays Laboratory Objectives

• 1. To prepare competent
• bacteria
• 2. To perform a transformation of
• competent bacteria with
• plasmid DNA
• 3. To quantify transformation
• efficiency

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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
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Transformation of chemically competent bacteria

• Incubation with high concentrations of cold
  calcium chloride followed by a brief heat shock

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Other DNA delivery methods Electroporation

• Electric shock opens transient pores in cell
• membrane

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Other DNA delivery methods Microprojectile
bombardment

• Shoots projectiles of gold or tungsten
  coated with DNA or RNA into cells
• Generally used with eukaryotic cells

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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
• 3. Gene of interest

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Map of Parent Vector pMAB58
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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.
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Propagation of Clonal Isolates
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Controls

• Control tests the competency of cells
• - Control tests the efficacy of selectable
  marker

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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)