bacterial genomics chapter 10, page 251 - mcccblinderl/documents/unit3students09.pdfbacterial...
TRANSCRIPT
1
Bacterial Genomics Chapter 10, page 251
First sequenced was Haemophilus influenzae
1.83 million bp
2
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
3
Single circular chromosome
http://www.sinauer.com/cooper/4e/micrographs0603.html
http://www.emc.maricopa.edu/faculty/farabee/biobk/bactchromo.gif
4
E. coli biology
Prokaryote
nucleoid region contains the chromosome
Neisseria gonorrhoeae.
5
E. coli reproduction
Bacteria reproduce by binary
fission -> Exponential growth
6
Bacterial growth
colony - visible cluster of
clones
about 1 million cells /colony
lawn – entire plate is covered,
no individual colonies visible
Growth on agar plate
7
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
8
Bacterial growth curve
9
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
10
Solid and liquid culture
Growth in liquid media Growth on agar plate
11
Phenotypes
Prototroph
can synthesize requirements from minimal
media
Auxotroph
nutritional mutant
Requires one or more supplements to grow
12
Bacterial phenotypes
Resistant to ampicillin = Ampr
Sensitivity to streptomycin = Strs
auxotroph mutant requires tryptophan = Trp-
trp-leu-thi+tetr ?
13
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
quorum sensing
http://www.pbs.org/wgbh/nova/sciencenow/34
01/04.html
14
15
How do bacteria undergo genetic
recombination?
16
Conjugation
parasexual mating
one-way transfer of genetic information from
“male” to “female” bacteria
17
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- leu-
OBTAIN ---> a few prototrophs that grow on
minimal media:
What would the genotype of this prototroph be?
18
Fig 18.2
Its rare ! 1 /10,000,000
Genetic recombination
19
Fig. 18.3 Davis U-tube showed that
conjugation requires cell/cell contact
met- bio- thr- leu-
Media can pass but cells can’t no prototrophs obtained
Show that cell-cell contact is required
filterNote the filter
20
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+
21
Conjugation fig. 18.5
F+ + F- = 2F+
Steps: Pilus -> nick DNA -> transfer DNA ->
make double stranded -> break pilus
22
F factor is a plasmid
94,000 bp
Must have an origin of replication (ori) to be maintained
can transfer to other cells.
()
23
24
Recombination (rare): Integration of F factor into
chromosome
Hfr strain fig. 18.5
25
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 F-
26
genetic recombination with Hfr
The transferred DNA may degrade or undergo homologous recombination
27
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
28
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
Hfr strains allow mapping of the E.
coli chromosome!
29
Lederberg’s experiment explained
fig. 18.7
30
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.
Elie Wollman & François Jacob
31
The mating:
Hfr H (aziRtonRlac+gal+strS)
x
F- (aziStonSlac-gal-strR)
32
Fig. 18.7
33
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)
34
35
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 2: AXPTM
STRAIN 3: BNCAX 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?
36
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)
37
Filter prevents cell contact,
transduction still occurs
38
Viral infection
1. Virus adsorbs to cell and injects DNA
39
40
2. normal bacterial activity is shut down
and bacterium becomes a “phage
factory”
41
42
3. host DNA broken into pieces, new
viruses released to infect new cells
43
chromosomal DNA is chopped as
viruses destroy cell
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
49
Bacteriophage phenotypes
virulent phage - always lytic, cannot
become a prophage
temperate phage - lysogenic
Temperate phage
50
51
Transformation
Naked DNA enters bacterial cell. Brings new
genes
Plasmids are extrachromosomally maintained
52
Plasmids are cloning vectors
(ch 8 pg 179)
pUC19 plasmid, a cloning vector
ampr gene
ori
restriction sites
(multiple cloning site)
53
Ampr
Ori
araC
GFP
54
Transformation in the laboratory
Make cells competent by calcium chloride
42 degree C heat shock facilitates uptake
Both have ori
55E. Coli genome is 4.6 million bp pGlO is 5,371 bp
genome
Bla gene encodes ampicillin
resistance
56
Ampicillin antibiotic inhibits cell wall
synthesis and cell lyse
Bla gene encodes beta lactamase which
breaks down ampicillin
57
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)
58
Aequorea victoria – source of the GFP gene
59
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
60
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
61
GFP gene cloned into plants
Arabidopsis thaliana
seedlings
Reporter gene
62
C. elegans
GFP a reporter for olfactory receptor gene
expressed when worms sense the odorant,
diacetyl
63
M. musculus (mouse)
GFP reporter for MHC
gene
64
GFP embryoGFP mother with GFP-
minus embryo
65
Every cell has GFP
66
Anopheles gambiae cells GFP and the reaper
apoptosis gene
67
Hoxc13-GFP fusion protein expression in
nails of embryonic day 14.5 mouse
Brain tumor expressing rfp
GFP and YFP reporter for stem
cells
69
Glow fish pets
70
71
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
Operon
group of coordinately regulated genes
One promoter for a number of genes
Polycistronic mRNA
1 mRNA molecule has info from multiple
genes
72
73
E. Coli Lac Operon
E. coli cells can convert lactose to glucose and
galactose
74
The Lac Operon allows for
coordinate gene expression
Note: 1 mRNA, promoter
75
3 STUCTURAL GENES = Z, Y, A
Lac Z gene encodes b-galactosidase enzyme
b-gal
lactose ------------- glucose + galactose
76
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)
77
Fig. 19.2 hydrolysis
This only occurs in the presence of
lactose, the inducer
78
Proteins ->
DNA ->
promoter = regulates transcription of ZYA
operator = must be unbound for P to be “open”
79
REPRESSOR PROTEIN (I)
Encoded by Lac I gene
Binds to operator
Prevents RNA pol from binding to promoter
80
Is this operon ON or OFF?
Is lactose PRESENT or ABSENT?
Lac I, P, O, ZYA genes are CIS elements
81
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
82
Lactose (the inducer) enters the cell
Binds repressor protein causing a conformational change
•
83
repressor binds to operator
polymerase cannot bind promoter
no transcription of ZYA genes
No lactose:
84
NO LACTOSE
86
Operon mutants
Mutant Mutant Phenotype
lac I- constitutive expression because…
Oc constitutive expression because …
P- no expression of operon because …
lac Z- ?
87
Operon on, or off in the absence of
lactose? Presence of lactose?
Lac I- (I- P+O+Z+Y+A+)
Lac Oc (I+P+OcZ+Y+A+)
88
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
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
Jellyfish DNA
GFP gene
Recombinant plasmid
pGLO
cut plasmid
plasmid
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
1. Isolate (normal) human DNA
2. Cut hGH gene out with restriction enzymes
….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 1987
Factor VIII for hemophiliacs 1993
Interferon for chemotherapy 1993
EPO for anemia 1992
FSH for fertility clinics 1996
TPA to prevent blood clots 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
Screening a library fig.
8.11
Each colony contains a
different fragment of DNA
They are unordered!
Need many plates!
cDNA library (coding regions only) = made
from mRNA
Tissue specific library
Chromosome specific library
Caveats
1. Restriction enzymes may cut within genes
2. Need vast numbers of recombinant bacteria
to represent entire genome
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
Alcohol dehydrogenaseLane 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
104Northern blot to assay mRNA levels in various tissues
cDNA library:Each tissue type expresses different genes
Chromosome specific library
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 increased seed costs
Decreased use of pesticides pesticide resistant bugs
Decreased use of herbicides resistant weeds
Increased nutrition new allergens
Increased shelf life may spread to other plants
Can remove allergens 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
1. Isolate human EPO gene (for rbc production)
2. Ligate to tissue-specific promoter
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)
The fish that has been mainly caught in the
nets of criticism is an Atlantic salmon with a
growth hormone gene from Chinook
salmon.
Researchers in Cuba and the UK have
engineered tilapia to grow and put on
weight up to 300% faster
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
1. Obtain somatic cell from donor ewe 2. Serum starve to induce Go
3. Place nucleus into enucleate egg
4. Grow for 6 days in lab
5. Implant into surrogate mother
277 embryos -> 1 lamb (Dolly)
http://learn.genetics.utah.edu/units/cloning/
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/cloningornot/
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 light brown = flexibleYellow = no federal government funding