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
3
http://www.emc.maricopa.edu/faculty/farabee/biobk/bactchromo.gif
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
Growth on agar plate
 about 1 million cells /colony

 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- leu-
OBTAIN --->
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 F-
25
 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)

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 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?
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 mother with GFP-
GFP embryo minus 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 …
P- no expression of operon because …
lac Z- ?

86
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+)

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
Jellyfish DNA plasmid

GFP gene

cut plasmid

Recombinant plasmid Next

pGLO 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?
26 inches tall
 Today
 hGH has been cloned
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
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
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
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 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)

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

3. Place nucleus into enucleate egg

4. Grow for 6 days in lab
5. Implant into surrogate mother

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 light brown =

flexible
Yellow = no federal government funding