Download Bacterial recombination

Bacterial Genomics
Chapter 10, page 251
First sequenced was
Haemophilus influenzae
 1.83 million bp
1
Escherichia coli (1997)
 Located in the lower intestines of animals
 Pathogenic strains (ex. E. coli 0157)
 Genome


4.6 megabases
~ 4000 genes, ~88 % of genome open reading
frames
2
Single circular chromosome
http://www.sinauer.com/cooper/4e/micrographs0603.html
http://www.emc.maricopa.edu/faculty/farabee/biobk/bactchromo.gif
3
E. coli biology
 Prokaryote
 nucleoid region contains the chromosome
Neisseria gonorrhoeae.
4
E. coli reproduction
 Bacteria reproduce by binary
fission -> Exponential growth
5
Bacterial growth
 colony - visible cluster of
clones

about 1 million cells /colony
Growth on agar plate
 lawn – entire plate is covered,
no individual colonies visible
6
Growth of bacteria (E. coli)
 Lag phase - slow or no apparent growth
 Log phase –double every 20’ to 1 X 109/ml
 Stationary phase
nutrient and/or oxygen limited
 Cell number remains constant
 Death phase


Nutrients gone, toxic products build up, cells die
7
Bacterial growth curve
8
Growth media
 minimal media =only essentials
provided


Sugar (carbon source) + salts
bacteria synthesize aa, nucleotides,
vitamins
 complete media
 selective media

Allows one species to grow while
selecting against another
9
Solid and liquid culture
Growth in liquid media
Growth on agar plate
10
Phenotypes
 Prototroph

can synthesize requirements from minimal
media
 Auxotroph


nutritional mutant
Requires one or more supplements to grow
11
Bacterial phenotypes
 Resistant to ampicillin = Ampr
 Sensitivity to streptomycin =
Strs
 auxotroph mutant requires tryptophan = Trp-
trp-leu-thi+tetr ?
12
Bacterial mutants
 Nutritional mutants

Auxotrophs that require supplement to grow
 Conditional mutants

The mutation is only expressed in a certain
condition
 Resistance mutant

Antibiotic resistance in bacteria
13
 quorum sensing
 http://www.pbs.org/wgbh/nova/sciencenow/34
01/04.html
14
How do bacteria undergo genetic
recombination?
15
Conjugation
 parasexual mating

one-way transfer of genetic information from
“male” to “female” bacteria
16
E. coli nutritional mutants
demonstrate conjugation
Mix auxotrophs – alone cannot grow on
minimal media:
Strain A met- bio- thr+ leu+
Strain B met+ bio+ thr- leuOBTAIN --->
a few prototrophs that grow on
minimal media:
What would the genotype of this prototroph be?
17
Fig 18.2
Its rare ! 1 /10,000,000
Genetic recombination
18
Fig. 18.3 Davis U-tube  showed that
conjugation requires cell/cell contact
met- bio-
thr- leu-
filter
Note the filter
Media can pass but cells can’t  no prototrophs obtained
Show that cell-cell contact is required
19
F factor (plasmid) carries DNA
from “male” to “female” bacterium
 F factor
 circular,
episomally maintained piece of
DNA
 Encodes

F pilus on donor cell
Donor cell is F+
20
Conjugation fig. 18.5
F+
+
F-
=
2F+
Steps: Pilus -> nick DNA -> transfer DNA ->
make double stranded -> break pilus
21
F factor is a plasmid
 94,000 bp
 Must have an origin of replication (ori) to be
maintained
 can transfer to other cells.
()
22
23
Recombination (rare): Integration of F factor into
chromosome
Hfr strain fig. 18.5
24
Hfr conjugation: F factor would transfer last
• The first DNA to be transferred is the
chromosomal DNA
• Pilus is broken before F factor is transferred
• Recipient cell remains F25
genetic recombination with Hfr
The transferred DNA may degrade or undergo homologous recombination
26
Comparing an Hfr to F+ strain
F+ x F-
recipients are F+
Low frequency of recombinants upon
conjugation
Hfr x F-
recipients are F-
High frequency of recombinants upon
conjugation
27
Hfr strains allow mapping of the E.
coli chromosome!
 Site of integration and orientation of plasmid
integration in the Hfr bacterial DNA is random
 Linear transfer of genes
 So, the time it takes for a particular gene to
transfer depends on where its located on the
chromosome
28
Lederberg’s experiment explained
fig. 18.7
29
Interrupted mating technique to
map genes on E. coli
1. Mix donor and recipient cells.
Hfr strs
+ F- strr
2. Incubate to allow conjugation to get started
3. At time t, blend the culture in the kitchen blender. This
disrupts the cell pairs but does not break the individual
cells.
4. Plate recipient cells (use streptomycin selection – why?).
5. Screen for recombinant markers.
30
Elie Wollman & François Jacob
The mating:
Hfr H (aziRtonRlac+gal+strS)
x
F-
(aziStonSlac-gal-strR)
31
Fig. 18.7
32
E. coli minute map = 4.7 million bp
(4377 genes)
Clock face.... Gene controlling
Noon+
threonine synthesis
1 o'clock
lactose degradation (lac-operon)
2 o'clock
galactose -> glucose (gal-operon)
3 o'clock
tryptophan synthesis (trp-operon)
5 o'clock
histidine synthesis (his-operon)
7 o'clock
lysine synthesis
8 o'clock
streptomycin resistance
9 o'clock
mannitol degradation
10 o'clock
Place where chomosome synthesis begins in both
directions ("OriC")
11 o'clock
methionine synthesis
Noon"F"-episome (where "F" is inserted)
33
34
Map genes using different Hfr strains
In E. coli, four Hfr strains donate the genetic markers
shown in the order given:
STRAIN 1: QWDMT
STRAIN 3: BNCAX
STRAIN 2: AXPTM
STRAIN 4: BQWDM
What is the order of these markers on the circular
chromosome of the original F+?
What is the location and orientation of the F factor
integration in the bacterial chromosome?
35
Transduction –phage mediated transfer
of genes into bacteria
 Phage – a virus that infects bacteria
 Salmonella typhimurium bacteria and P22 virus
 U-tube
experiment

mix 2 auxotrophs

prototrophs appear (low rate)
36
 Filter prevents cell contact,
transduction still occurs
37
Viral infection
1. Virus adsorbs to cell and injects DNA
38
39
2. normal bacterial activity is shut down
and bacterium becomes a “phage
factory”
40
41
3. host DNA broken into pieces, new
viruses released to infect new cells
42
chromosomal DNA is chopped as
viruses destroy cell
43
Faulty head stuffing
 As chromosomal DNA is broken, a piece can
get packaged into a virus.
 This virus can infect a new cell and transfer
genes from the first bacterium
44
Gene therapy with virus
Ch 9, pg 231
 Objective : insert a normal gene into human
DNA that contains mutant gene
 Use virus as vector
45
 Remove viral replication genes (can no
longer cause disease)
 insert human gene
 Infect the human with the engineered
virus
 The virus is the delivery system
46
Gene Therapy ADA 1990
 Genetic defect in white blood cells
 Severe immune deficiency
47
 Remove wbc
 Engineer in lab
 Infuse into patient
 Repeat
48
Bacteriophage phenotypes
 virulent phage - always lytic, cannot
become a prophage
 temperate phage - lysogenic
49
Temperate phage
50
Transformation

Naked DNA enters bacterial cell. Brings new
genes

Plasmids are extrachromosomally maintained
51
Plasmids are cloning vectors
(ch 8 pg 179)
 pUC19 plasmid, a cloning vector
ampr gene
ori
restriction sites
(multiple cloning site)
52
Ampr
Ori
araC
GFP
53
Transformation in the laboratory


Make cells competent by calcium chloride
42 degree C heat shock facilitates uptake
54
Both have ori
genome
E. Coli genome is 4.6 million bp
pGlO is 5,371 55bp
Bla gene encodes ampicillin
resistance
Ampicillin antibiotic inhibits cell wall
synthesis and cell lyse
Bla gene encodes beta lactamase which
breaks down ampicillin
56
LABORATORY
 E. coli bacteria strain K12/HB101
 Non pathogenic
 Grows well at room T or 37oC
 Prototroph grows in Luria broth (LB)
 Host for plasmid DNA
 Killed by ampicillin (if untransformed)
57
 Aequorea victoria – source of the GFP gene
58
Engineering the plasmid, pGLO
1. Isolate jellyfish DNA
2. Use restriction enzymes to
cut out GFP gene
3. Purify GFP gene
4. Ligate GFP into plasmid
59
pGLO plasmid
ori –replication of plasmid
Ampr (bla)- ampicillin resistance
Only transformed bacteria can grow
in presence of amp
GFP gene
Ara C GFP gene expressed in presence of
arabinose sugar
60
GFP gene cloned into plants
Arabidopsis thaliana
seedlings
Reporter gene
61
C. elegans
 GFP a reporter for olfactory receptor gene
 expressed when worms sense the odorant,
diacetyl
62
M. musculus (mouse)
GFP reporter for MHC
gene
63
GFP embryo
GFP mother with GFPminus embryo
64
Every cell has GFP
65
Anopheles gambiae cells GFP and the reaper
apoptosis gene
66
 Hoxc13-GFP fusion protein expression in
nails of embryonic day 14.5 mouse
67
Brain tumor expressing rfp
GFP and YFP reporter for stem
cells
69
Glow fish pets
70
The Lac Operon 1961, Jacob and Monod
E. coli and other bacteria
 Bacterial Genes

Many genes are constitutively expressed


these are “housekeeping” genes
Other genes are regulated

Can be turned on, or off depending on cell needs
71
Operon
 group of coordinately regulated genes
 One promoter for a number of genes
 Polycistronic mRNA
 1 mRNA molecule has info from multiple
genes
72
E. Coli
Lac Operon
 E. coli cells can convert lactose to glucose and
galactose
73
The Lac Operon allows for
coordinate gene expression
Note: 1 mRNA, promoter
74
3 STUCTURAL GENES = Z, Y, A
Lac Z
gene encodes b-galactosidase enzyme
b-gal
lactose ------------- glucose + galactose
75
LacZ gene is only transcribed when lactose
sugar is present

b- gal is an inducible enzyme
(induced by lactose from 5 copies enzyme to 1000s)
76
This only occurs in the presence of
lactose, the inducer
Fig. 19.2 hydrolysis
77
DNA ->
Proteins ->
promoter = regulates transcription of ZYA
operator = must be unbound for P to be “open”
78
REPRESSOR PROTEIN (I)
 Encoded by
Lac I gene
 Binds to operator
 Prevents RNA pol from binding to promoter
79
Is this operon ON or OFF?
Is lactose PRESENT or ABSENT?
Lac I, P, O, ZYA genes are CIS elements
80
INDUCER (LACTOSE SUGAR)
 LACTOSE PRESENT
•
•
•
•
•
Lactose enters
Binds repressor protein (I) causing a
conformational change
This pulls the repressor off the operator
RNA polymerase transcribes genes
Cell metabolizes lactose
81
Lactose (the inducer) enters the cell
Binds repressor protein causing a conformational change
•
82
No lactose:
repressor binds to operator
polymerase cannot bind promoter
no transcription of ZYA genes
83
 NO LACTOSE
84
 Lac operon animation
85
Operon mutants
Mutant
Mutant Phenotype
lac I-
constitutive expression because…
Oc
constitutive expression because …
Plac Z-
no expression of operon because …
?
86
Operon on, or off in the absence of
lactose? Presence of lactose?
Lac ILac Oc
(I- P+O+Z+Y+A+)
(I+P+OcZ+Y+A+)
87
 Remember, repressor and polymerase are
proteins which are diffusible


These proteins bind DNA
They act in TRANS
 The promoter, operator, and ZYA and I are
genes and cannot move

They act in CIS
88
Gene cloning in bacteria
1. Isolate DNA from organism
2. Cut DNA and vector with restriction enzyme(s) to
produce overhangs (sticky ends)
3. Ligate to form recombinant DNA
4. Transform bacteria with engineered vector

use selectable marker
5. Grow bacteria
6. Isolate protein from bacteria
plasmid
Jellyfish DNA
GFP gene
cut plasmid
Recombinant plasmid
pGLO
Next
transformation
 purify GFP
 Crack open the E. coli cells -- purify GFP
Bacterial chromosome
plasmid
Pure GFP protein
Human Growth Hormone (hGH) cloned into
bacteria (1980s)
 hGH is a 191 aa peptide produced by
the pituitary gland
 Pre-1980s hGH purified from
cadaver brains
Drawbacks?
 Today

hGH has been cloned
26 inches tall
Isolate (normal) human DNA
2. Cut hGH gene out with restriction enzymes
1.
….ggattgcgtacgctttgatcgtagtaataggacctagtgtgtacgtaagc
gg……
….ccattcgcatgcgaaagtagcatcattatccaggatctcacatgcattcg
cc……
pure hGH gene
gatcgtagtaatagg
agtagcatcattatcc
3. Ligate hGH gene into plasmid vector = recombinant
DNA
4. Transform bacteria
5. Grow bacteria - billions of copies overnight
bacteria will express the hGH gene to make hGH
protein
6. Purify hGH, bottle, sell, and inject before puberty
 Reported effects include decreased body fat,
increased muscle mass, increased bone
density, increased energy levels, improved
skin tone and texture, and improved immune
system function.
 Banned by IOC and NCAA (and others)
Other cloned drugs made by bacteria
Human insulin
Factor VIII for hemophiliacs
Interferon for chemotherapy
EPO for anemia
FSH for fertility clinics
TPA to prevent blood clots
1987
1993
1993
1992
1996
1996
Many drugs for domestic animals
cancer, arthritis, emphysema and other drugs
Vaccines (hepatitis B surface antigen)
Gene library
 Genomic library = Collection of clones that
contain the entire genome
 Need > 50,000 bacterial clones to hold the
entire human genome?
Getting a gene into bacteria
 *Transformation (plasmid)
 Infection (phage)
 Gene gun
Each colony contains a
different fragment of DNA
They are unordered!
Need many plates!
Screening a library fig.
8.11
 cDNA library (coding regions only) = made
from mRNA

Tissue specific library
 Chromosome specific library
Caveats
Restriction enzymes may cut within genes
2. Need vast numbers of recombinant bacteria
to represent entire genome
1.
DNA libraries – Ch. 8
 Genomic library– how many cells are needed
to represent the entire human genome (46
chromosomes, 3 billion nucleotide base
pairs)?
 cDNA library – what type of genes would be
represented in a cDNA library of stomach
lining?
 Chromosome specific library – how many
chromosome libraries are required to
represent the entire human genome?
Genomic: Need many bacterial transformants to
represent human genome
http://www.accessexcellence.org/RC/VL/GG/ecb/ecb_images/10_23_genomic_library.jpg
cDNA library:Each tissue type expresses different genes
Alcohol dehydrogenase
Lane 1 RNA marker
Lane 2 total RNA (Liver)
Lane 3 Brain
Lane 4 Cerebellum
Lane 5 Cerebrum
Lane 6 Kidney
Lane 7 Liver
Lane 8 Lung
Lane 9 Spleen
Lane 10 Thymus
Lane 11 Testis
Northern blot to assay mRNA levels in various tissues104
Chromosome specific library
 Lac operon animation
Cloning into plants (GM) Ch. 9
 Transgenic plants

Plants that acquire a new genetic trait by
direct introduction of gene
 Inject gene into plant embryo (or plasmids
can be used)
How to make a transgenic plant
Isolate non- edible (wild) tomato plant DNA
 Cut DNA with restriction enzyme
 Cut out the gene that encodes sweetness
(restriction enzyme)
 Cut plasmid (Ti)with same enzyme
 Ligate to form rDNA
 transform other plant embryo  tissue
culture
How to make a transgenic tomato
Benefits
Drawbacks
 Increased crop yield
 Resistance to drought, freezing
 Decreased use of pesticides
 Decreased use of herbicides
 Increased nutrition
 Increased shelf life
 Can remove allergens
increased seed costs
pesticide resistant bugs
resistant weeds
new allergens
may spread to other plants
harmful to insects?
Bt corn
 Corn plant engineered with gene
that codes for a protein lethal to
the corn borer
The corn root worm
Golden Rice
 Many in world are deficient in Vitamin A

Leading cause of childhood blindness
(500,000 new cases per year)
 Rice engineered to produce vitamin A!
Controversial…….
 Do we need legislation for labeling of GM foods?
 Should GM genes, plants, animals, be
patented?
Cloning genes into animals
 A transgenic animal carries a foreign gene
deliberately inserted into its genome.
Transgenic goats Ch. 9
Produce human protein
(drug) in milk
Pharming
Transgenic animals to produce human
protein in milk
Isolate human EPO gene (for rbc production)
2. Ligate to tissue-specific promoter
1.

Promoter ONLY active in mammary gland 
protein only made in milk
1. Inject gene construct into animal
embryo (fertilized egg)
2. Implant embryo into
surrogate mother -> kid
is born
 How do we know if kid
is transgenic?
 How can we get the
transgenic kid to
produce human drug?
3. Easy to purify from milk
• One herd can supply the world’s need of a
particular drug (protein)
• Clean, disease free
Pail of milk with EPO
Bottled EPO drug
 Transgenic
animal
contains one
foreign gene
Other proteins made in transgenic
sheep and goat milk
• Spider silk (BioSteel)
– The dragline form of spider silk is regarded as the
strongest material known; it's 5 times stronger than
steel and twice as strong as Kevlar.
genus Araneus
• 2009: FDA Approves Drug From Transgenic Goat
Milk ATryn, human antithrombin protein
• Anti HIV protein
• Anticancer drugs
• Alpha1-antitrypsin for emphysema
Mouse model organism
 These mice are
models for human
disease (Alzheimer)
 This mouse is
genetically modified
to be diabetic
Knockout mice
 Normal gene (in embryo) has been replaced
with non-functional gene
 Examples



Cystic Fibrosis (CF) - The Cftr knockout
mouse
Cancer - p53 knockout mouse has a disabled
Trp53 tumor suppressor gene
Glaucoma - The DBA/2J mouse exhibits many
of the symptoms that are often associated with
human glaucoma
Agriculture
 This pig is genetically engineered to be able
to digest more and produce less manure
 Other pigs produce meat high in omega 3
fatty acids
Medicine
 This chicken produces a human antibody in
her eggs
Xenotransplantation
 Pigs have similar sized organs to humans
 May involve knocking out pig cell surface
antigens
to prevent hyperacute rejections
Fish farming
 genetically engineered salmon grow faster (not FDA
approved)
Researchers in Cuba and the UK have
engineered tilapia to grow and put on
weight up to 300% faster
The fish that has been mainly caught in the
nets of criticism is an Atlantic salmon with a
growth hormone gene from Chinook
salmon.
Patenting
 Raw products of nature are not patentable.
 DNA products become patentable when they
have been isolated, purified, or modified to
produce a unique form not found in nature.
 Millions of patents
3 types of cloning
 1.
gene cloning
Recombinant bacteria (as in lab)
 Transgenic plants
 Transgenic animals

 2. reproductive cloning
 Yields an organism
 Embryo twinning or nuclear transfer
 3. therapeutic cloning
 nuclear transfer for stem cells to treat
disease
Reproductive cloning
Embryo twinning

1 sperm + 1 egg - 2 embryos (genetically
identical)
 http://learn.genetics.utah.edu/units/cloning/wh
atiscloning/
Nuclear transfer method - The clone’s
DNA is a genetic copy of the donor
SCNT =
somatic cell
nuclear
transfer
pg. 577
1997 Ian Wilmut
http://learn.genetics.utah.edu/units/cloning/
1.
Obtain somatic cell from donor ewe
2.
Serum starve to induce Go
Place nucleus into enucleate egg
4. Grow for 6 days in lab
5. Implant into surrogate mother
3.
277 embryos -> 1 lamb (Dolly)
 Somatic cell nuclear transfer
Our somatic nuclei (DNA
from a differentiated cell)
can be reprogrammed to
embryonic state!
Cloning game
http://learn.genetics.utah.edu/units/cloning/cloni
ngornot/
Why clone animals?
 Models for disease
 Pharming
 Endangered species – ex. Mouflon sheep,
the surrogate mother was a domestic sheep!
 Reproduce deceased pet
 Help infertile couples
 $1395 PetBank provides pet lovers with the option to
clone their exceptional pets. If you don't save your pet's
DNA today, cloning may not be possible tomorrow.
Saving your pet's DNA is simple, safe and lasts forever.

$150 annual storage fee after the first year.
 K.C., the first
animal produced
by cloning from a
cell taken from a
carcass, was born
in April 2002.
 Yoda and Sue were
cloned from a
Landrace boar in
2002.
 "FDA: Food from animal
clones safe to eat"
Associated Press, October
31, 2003
 A Boca Raton, Florida, couple paid a California firm $155,000
to clone their beloved Labrador retriever, who died from cancer
a year ago. The clone, a 10-week-old puppy dubbed Lancey,
was hand-delivered to them earlier this week by Lou
Hawthorne, chairman of BioArts International, a biotechnology
company.
 .
Problems with reproductive cloning
 High failure rate < 3% success rate





2003 first horse cloned (Prometea) 22 embryos, 800
eggs
Enucleate egg may not function
Embryo may not divide
Embryo may not implant
Miscarriage
 Large offspring syndrome (LOS)
 With abnormally large organs that don’t
function correctly
 Abnormal gene expression
 We don’t understand how the nucleus is
reprogrammed (its old DNA in a new egg!)
 Telomere problems
 Older DNA has shortened telomeres, but
some clones show lengthened telomeres
Ethical implications
 Is human cloning "playing with nature?" If so,
how does that compare with other reproductive
technologies such as in vitro fertilization or
hormone treatments?
 If a clone originates from an existing person,
who is the parent?
 What are some of the social challenges a
cloned child might face?
 Should cloning research be regulated? How,
and by whom?
All countries have banned human
reproductive cloning.
Dark brown = permissive policy
flexible
Yellow = no federal government funding
light brown =