Bacterial Transformation
Broad and Long Term Objective
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.
2.
3.
To prepare competent cells
To perform a plasmid transformation
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.
2.
3.
Mode of Delivery
Selectable Marker
Propagation of Clonal Isolates
Nucleic Acid Delivery Methods
1.
2.
3.
Chemical treatment
Electroporation
Microparticle Gun
Chemical Treatment
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
Generally used with Eucaryotes
Selectable Marker
1.
2.
Antibiotic Resistance Markers
Complementation using Essential Metabolic Enzymes
Map of Positive Control Vector
Map of Parent Vector pMAB58 of
Experimental cDNA Clone
SnaBI
BspEI
SwaI
PpuMI
XbaI
Bsu36I
ARS4/Cen6
TRP1
7000
DraIII
Amp
AhdI
1000
F1
6000
BamHI
pT7
7577 bps
Ter ADH
attB2
pMAB58
2000
ori
5000
ccdB
3000
SacII
++ AatII
4000
attB1
BsrGI
MluI
AD
NLS
NotI
pADH
EcoRI
BsaBI
SmaI
BsrGI
BsmI
XbaI
EcoRI
PacI
MluI
BsrGI
SexAI
BseRI
Eco47III
RsrII
NspV
BsaBI
Asp718I
KpnI
ApaI
Bsp120I
StyI
BsmI
AlwNI
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
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.
2.
3.
Perform a Plasmid Miniprep
Quantitate Plastmid Isolated
Determine the Size of the Plasmid