Lecture 7 Guide

Lecture 7 Guide -...

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Genetics
of
Bacteria
and
their
Viruses
 I.
Bacterial
genetics
 
 
 
 
 
 
 
 
 
 
 

A.

Introduction
 
1.
Power
in
numbers
 
2.
Growth
of
bacteria
and
nomenclature
 B.
Conjugation
 
1.
F,

F‐
 
2.
Hfr,
F’
 C.
Transformation
 D.
Bacteriophage
and
Transduction
 
1.
Lytic,
lysogenic
 
2.
rII
locus
 II.
Foundations
of
recombinant
DNA
technology
 
 
 
 
 
 
 A.
Bacterial
restriction
enzymes
 B.
Constructing
recombinant
DNA
 
1.
Donor,
vector
 
2.
Libraries
and
screening
 C.
Restriction
mapping
 

 
 
 
Genetics
of
Bacteria
and
their
Viruses

 Objectives:
 Be
able
to
determine
gene
order
and
map
distance
in
bacterial
chromosomes
 Learn
how
recombinant
DNA
libraries
are
made
and
screened
 Be
able
to
produce
and
analyze
restriction
maps
of
DNA,
and
work
with
RFLP
data
 Vocabulary:
 complete
media,
minimal
media
 conjugation
 exconjugant
 F
factor
 plasmid
 F’
 Hfr
 partial
diploid
 donor,
recipient
 minutes
of
transfer
 transformation,
transformant
 transduction,
transductant
 lytic
 lysogenic
 colony
 plaque
 agar
plates
 replica
plating
 restriction
endonuclease
enzymes
 recombinant
DNA
vectors
and
donor
 restriction
maps
and
mapping
 RFLP
 Bacterial colonies, each derived from a single cell Replica plating Minimal medium Bacteria exchange DNA by several processes Bacteria conjugate by using pili Figure
5‐7
 Two types of DNA transfer can take place during conjugation A single crossover inserts F at a specific locus, which then determines the order of gene transfer Tracking time of marker entry generates a chromosome map Figure
5‐12b
 Tracking time of marker entry generates a chromosome map Figure
5‐12a
 Use
this
figure
as
a
sample
mapping
problem.
 1.
From
the
information
in
part
a,
draw
the
 
gene
order,
F
placement
and
direction
of
transfer.
 2.
Check
your
answer
compared
to
part
b.
 Crossovers integrate parts of the transferred donor fragment Figure
5‐11
 Faulty outlooping produces F´, an F plasmid that contains chromosomal DNA A plasmid with segments from many former bacterial hosts Figure
5‐19
 Transformation: Mechanism of DNA uptake by bacteria Recall
early
experiments
 on
transformation
and
DNA:

 Griffiths;
 Avery,
Macleod
and

 McCarty
 Electron micrograph of phage infection Figure
5‐24
 Cycle of a phage that lyses the host cells Figure
5‐25
 Generalized transduction by random incorporation of bacterial DNA into phage heads From high cotransduction frequencies, close linkage is inferred λ phage inserts by a crossover at a specific site Figure
5‐32
 Bacteriophage
Genetics:
rII
locus
 Bacteria
and
Phage:


Foundations
of
Recombinant
DNA
 Inserting a gene into a recombinant DNA plasmid How amplification works Figure
20‐4
 A plasmid vector, pUC18 Figure
20‐5
 Plasmid
cloning
vector
 pBluescript: popular plasmid for many years. The lacZ gene allowed for blue/white color screening for the presence of an insert during the cloning procedures, as shown on the agar plate below. The blue colonies represent unsuccessful cloning since the lacZ gene was not interrupted. Cloning in phage λ Figure
20‐6
 Modes of delivering recombinant DNA into bacterial cells Finding the clone of interest by using DNA or RNA probes Figure
20‐9
 Restriction
endonuclease
analysis
 Above: ethidium bromide stained agarose gel. M: molecular weight markers, in kilobase-paris, kb Above: lambda phage DNA digested with the restriction enzyme, HindIII. This is a commonly used molecular weight marker. Restriction
endonuclease
analysis
 Southern
Analysis

 Finding specific nucleic acids by using gel electrophoresis and blotting Southern
Analysis,
RFLP,
and
Disease

 Assignment

 Achieve
Objectives
List
(slide
2)
through
study
 and
practice.
 Solve
problems
from
textbook.

 Chapter
5:
 1,
2,
4,
8,
28,
29
 Chapter
20:
 3,
4,
6
 ...
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This note was uploaded on 09/26/2009 for the course BIS 98659 taught by Professor Kimbrell during the Summer '09 term at UC Davis.

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