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Requirements for a successful transformation
experiment:
introducing DNA into cells selectable marker on the introduced DNA with corresponding non-reverting alleles in the chromosome vector systems that allow propagation of cloned DNA in both Escherichio coliand yeast. TERM 2
Original method for yeast transformation
involved
DEFINITION 2 Incubating spheroplasted cells with DNA in the presence of polyethylene glycol (PEG) CaCl TERM 3
spheroplast
DEFINITION 3 A spheroplast is a cell from which the cell wall has been almost completely removed, as by the action of penicillin. TERM 4
polyethylene glycol
DEFINITION 4 Polyethylene glycol is a polyether compound with many applications from industrial manufacturing to medicine. TERM 5
More convenient and much more widely used
method involves
DEFINITION 5 treatment of cells with the alkali salt, lithium acetate (LiAc), followed by incubation with DNA and PEG
alkali salt
are salts which are the product of the neutralization of a strong base and a weak acid. TERM 7
lithium acetate
DEFINITION 7 Lithium acetate is used in the laboratory as buffer for gel electrophoresis of DNA and RNA. It has a lower electrical conductivity and can be run at higher speeds TERM 8
Electroporation
DEFINITION 8 is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. TERM 9
prototrophic
DEFINITION 9 cells are able to metabolize all necessary amino acids for survival. TERM 10
auxotrophic
DEFINITION 10 is most commonly defined as the inability of an organism to synthesize a particular organic compound required for its growth
Neomycin
ntibiotic produced from strains of the actinomycete Streptomyces fradiae Neomycin is an aminoglycoside antibiotic found in many topical medications such as creams, ointments, and eyedrops. TERM 17
ARS element
DEFINITION 17 contains the origin of replication in the yeast genome TERM 18
CEN element
DEFINITION 18 causes equal segregation of the products of replication into daughter cells TERM 19
AMP
DEFINITION 19 bacteria selectable marker usually AMP for resistance to ampicillin. TERM 20
Transformation
DEFINITION 20 Genetically changing cells through the administration of exogenous DNA
Transformation process are:
Uptake Incorporation (chromosomally, autonomously) Expression TERM 22
Successful of Transformation
DEFINITION 22 Introduction into cells Selectable marker Propagation TERM 23
Propagation
DEFINITION 23 Bacterial cloning and manipulation of the plasmid Stability and expression within the target cell TERM 24
Transformation Methods
DEFINITION 24 Polyethylene glycol (PEG) + CaCl2 with spheroplasts Lithium acetate + PEG + ssDNA Physical methods TERM 25
Physical methods
DEFINITION 25 Electroporation Mechanical (beads) Gene bombardment Bacterial conjugation
ARSs and Centromeres
Autonomously replicating sequence (ARS) Centromere sequences (CEN) TERM 32
Autonomously replicating sequence (ARS)
DEFINITION 32 Required for replication without homologous recombination TERM 33
Centromere sequences
(CEN)
DEFINITION 33 Encodes a sequence that will stick the plasmid to the mitotic spindle TERM 34
2 origin
DEFINITION 34 Naturally occurring mechanism Involves some tricky geneFcs Dependent on ippase This is all intenFonally vague TERM 35
Hypothesize
DEFINITION 35 Find out about the strains Find out about the plasmids Formulate hypotheses on what you expect
Reversion
Evolution favors mutations that restore important functions like metabolism. If your mutant strain is a point mutant, natural mutagenesis can revert it. TERM 37
Dominant Selection
DEFINITION 37 Using drug resistance as a method of selection Same concept dierent approach TERM 38
Using drug resistance as a method of
selection
DEFINITION 38 Neomycin Nourseothricin TERM 39
Controls
DEFINITION 39 Negative controls Positive controls TERM 40
Negative controls
DEFINITION 40 What eect does the procedure have on your outcome? Include everything but the experimental variable
CEN/ARS plasmid
is more stable than a simple ARS plasmid because the centromere mediates the attachment of the plasmid to the mitotic spindle, ensuring segregation to both mother and daughter cells. Because of the high fidelity of segregation, the copy number is maintained at one ortwo plasmids per cell. TERM 47
autonomously replicating plasmid that has
the 2mu origin of replication
DEFINITION 47 segregates in mitosis with about the same fidelity as a CEN plasmid, but is present at a much higher copy number, typically 20-50 copies per cell.
