bacterial transformation. broad and long term objective to characterize a single clone from an...

Bacterial TransformationBacterial Transformation

Broad and Long Term ObjectiveBroad and Long Term Objective

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

Research PlanResearch Plan

Preparation of Competent Cells and Bacterial Transformation

Growth of Transformant and Plasmid MiniPrep

Cycle Sequencing

Sequence analysis

Today’s Laboratory ObjectivesToday’s Laboratory Objectives

1.1. To prepare competent cellsTo prepare competent cells

2.2. To perform a plasmid transformationTo perform a plasmid transformation

3.3. To quantify transformation efficiencyTo quantify transformation efficiency

DefinitionsDefinitions

““Competency” refers to the Competency” refers to the abilityability to take up foreign DNA to take up foreign DNA

some bacterial cells are naturally competent and have special some bacterial cells are naturally competent and have special proteins that are involved in DNA uptake and integration into the proteins that are involved in DNA uptake and integration into the chromosomechromosome

artificially induced competent is where DNA uptake is induced by artificially induced competent is where DNA uptake is induced by chemical or physical meanschemical or physical means

Definitions Con’tDefinitions Con’t

““Transformation” is the Transformation” is the uptakeuptake of free foreign of free foreign DNA into the cellDNA into the cell

Components of a Components of a Transformation SystemTransformation System

1.1. Mode of DeliveryMode of Delivery

2.2. Selectable MarkerSelectable Marker

3.3. Propagation of Clonal IsolatesPropagation of Clonal Isolates

1.1. Chemical treatmentChemical treatment

2.2. ElectroporationElectroporation

3.3. Microparticle GunMicroparticle Gun

Nucleic Acid Delivery Methods

Chemical TreatmentChemical Treatment

Cold Shock with high concentrations of calcium chloride Cold Shock with high concentrations of calcium chloride and magnesium chlorideand magnesium chloride

ElectroporationElectroporation

Electric Shock Opens Pores in Cell WallElectric Shock Opens Pores in Cell Wall

Microparticle BombardmentMicroparticle Bombardment

Shoots projectiles of gold or tungsten coated with DNA or RNA into Shoots projectiles of gold or tungsten coated with DNA or RNA into cells using an inert gascells using an inert gas

Generally used with EucaryotesGenerally used with Eucaryotes

Selectable MarkerSelectable Marker

1.1. Antibiotic Resistance MarkersAntibiotic Resistance Markers

2.2. Complementation using Essential Metabolic Enzymes Complementation using Essential Metabolic Enzymes

Map of Positive Control VectorMap of Positive Control Vector

Map of Parent Vector pMAB58 of Map of Parent Vector pMAB58 of Experimental cDNA CloneExperimental cDNA Clone

pMAB58

7577 bps

1000

2000

3000

4000

5000

6000

7000

SwaIPpuMI

AhdI

AlwNI

Asp718IKpnIApaIBsp120I

StyIBsmI

PacIBsrGI

BsaBINspV

RsrIIEco47IIIBseRI

SexAIMluI

EcoRIXbaI

BsmIBsrGISmaI

BsaBIEcoRI

NotIMluI

BsrGIAatII++

SacII

BamHI

DraIII

Bsu36IXbaI

BspEISnaBI

ARS4/Cen6

Amp

ori

pADHNLS

ADattB1

ccdB

attB2

Ter ADH

pT7

F1

TRP1

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 TransformationPlasmid Transformation

ControlsControls

+ Control= tests the competency of cells+ Control= tests the competency of cells

- Control= tests the efficacy of selectable marker- Control= tests the efficacy of selectable marker

Transformation Efficiency:Transformation Efficiency:

Number of transformants/ng of DNANumber of transformants/ng of DNA Count number of colonies on plate after overnight growthCount number of colonies on plate after overnight growth Determine the proportion of the total transformation that was Determine the proportion of the total transformation that was

plated (ie, volume plated/total transformation reaction volume)plated (ie, volume plated/total transformation reaction volume) Express in terms of the amount of DNA used in the reactionExpress in terms of the amount of DNA used in the reaction

Number of transformants x (total transformation volume/volume plated)Number of transformants x (total transformation volume/volume plated)

ng of DNA used in transformationng of DNA used in transformation

Next WeekNext Week

1.1. Perform a Plasmid Miniprep Perform a Plasmid Miniprep

2.2. Quantitate Plastmid IsolatedQuantitate Plastmid Isolated

3.3. Determine the Size of the PlasmidDetermine the Size of the Plasmid