Download Bacterial recombination

Why do we care about homologous
recombination?
 Universal biological mechanism
 Bacteria can pick up new genes
 Biotechnology
 Gene knockouts in mice via homologous
recombination
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 DNA of interest in mouse chromosome
This is the gene targeted for replacement by an
engineered construct. Note flanking upstream and
downstream DNA sequences. The arrows pointing away
from the targeted gene represent the continuous
chromosomal DNA
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 1. Prepare construct DNA in lab with
selectable marker
Engineered construct used to replace the gene.
Upstream and downstream flanking DNA sequences
are identical to those which flank the targeted gene.
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 2. Add construct to embryonic stem cells
(ES) in culture
Amazingly, the DNA construct finds its way into ES
cell nucleus and aligns itself with targeted gene.
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 3. homologous recombination by cell
The chromosome now contains engineered construct in
place of the original allele. The original allele has been
recombined into the construct and is lost over time.
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4. Add ES cells to embryo  implant in
surrogate mother
5. Cross breed to create homozygous knockout
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Back to bacteria…..
 Hfr strains led to mapping of the E. coli
chromosome
 Interrupted mating technique to map genes
on E. coli
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Lederberg’s experiment explained
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Fig. 15.7
Hfr H (aziRtonRlac+gal+strS)
X
F-
(aziStonSlac-gal-strR)
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Circular chromosome
4.6 million bp (4.6 Mb)
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2. Transformation
 Naked DNA enters bacterial cell. Brings new
genes (can change bacteria phenotype)
 Bacterium with new DNA is a transformant
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Transformation (rare event)
 Natural flash animation
 Engineered

CaCl2 treat bacteria  competent cells

cell membrane permeable to naked DNA
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Plasmids can be cloning vectors
Ch 8 pg 175
 pUC19
ampr gene
ori
restriction sites
(multiple cloning site)
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Plasmid requirements in biotech
1. Ori for DNA replication
2. Selectable marker ex. ampr
1.
Only cells that take up the plasmid are resistant
to amp
3. Restriction enzyme sites
4. High copy number in E. coli (100/cell)
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1.Shimomura
Ampr
Ori
araC
GFP
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Viruses can bring new genes into a cell
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Transduction –phage mediated transfer
of genes into bacteria
 Bacteriophage – virus that infects bacteria

Lederberg and Zinder 1952
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phage
 DNA or RNA surrounded by protein coat
 genes encode for viral activity, viral parts
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Viral infection lytic cycle
1. Virus adsorbs to cell and injects DNA
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2. normal bacterial activity is shut down
and bacterium becomes a “phage
factory”
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3. host DNA broken into pieces, new
viruses released to infect new cells
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chromosomal DNA is chopped as
viruses destroy cell
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Generalized transduction
 A piece of chromosomal DNA gets packaged
into a virus = faulty head stuffing
 This transducing phage infects a new cell
and transfers genes from the first bacterium
 Homologous recombination occurs
 Flash animation generalized transduction
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Bacteriophage phenotypes
 virulent phage - always lytic, cannot
become a prophage
 temperate phage - lysogenic
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Temperate phage and lysogenic
pathway
 Phage DNA integrated
into specific location in
chromosome
 Prophage is lysogenic
 Phage gene represses
lytic cycle
 Flash animation
specialized transduction
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Growing phage
 Grow bacterial lawn on agar dish
 Add phage  infects bacteria
 Obtain plaques (where cells have lysed)
 Obtain phage lysate (contains phage)
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plaques
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Gene therapy with virus (Ch 10)
 Objective : insert normal gene into human DNA
 Candidates: people with single gene disorders
 Use virus as vector
Adenovirus. Child Health and
Human Development
Bioinformation video
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Gene Therapy ADA 1990
 Gene for adenosine deaminase


ADA normally eliminates deoxyadenosine
(from degraded DNA) (recessive disease)
dA toxic to lymphocytes
  Severe immune deficiency
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Ashanti Disilva was 4 and dying
1. remove viral replication genes
insert normal ADA gene into
virus
3. remove T cells from patient
4. infect cells with engineered
virus
5. infuse into patient
2.
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Problems with gene therapy
 Inflammatory response to virus  death
 Gene disrupts cell cycle gene cancer
 Other methods


Liposomes
Stem cells
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