genetic engineering

1. BACTERIAL GENETICS
Muhammad Bawa (PhD)

2. Key Words
• Genetics
• Bacterial genetics
• Mutation & its types
–
• Bacteriophage
• Mechanisms of gene
transfer
– Transformation
– Transduction
– Lysogenic conversion
– Conjugation

3. Bacterial Genetics
• Genetics is the study of heredity and variation.
The unit of heredity is gene, which is a segment of
DNA specifying for a particular polypeptide.
Introns - non coding sequences on a gene.
Exons - coding sequences on a gene translated into
gene products.
• Bacterial genetics is used as a model to understand DNA
replication, genetic characters, their changes & transfer
to next generations.

4. Nucleic Acids
• DNA ( deoxy ribonucleic acid ) : stores information
for protein synthesis.
• RNA ( ribonucleic acid ) : transcription & translation
of information for protein synthesis.
• Central Dogma : DNA RNA Protein

5. Structure Of DNA
• Proposed by Watson & Crick.
• Double helix model.
• Composed of 2 chains of polypeptides, each chain
has a backbone of deoxyribose sugar and phosphate
residues arranged alternately.
• 4 nitrogenous bases: Adenine (A) Purine
Guanine (G)
Thymine(T) Pyrimidine
Cytosine (C)

6. Double helical structure of
DNA by Watson & Crick

7. DNA
• DNA is copied by DNA polymerase
– In the 5  3 direction
– Initiated by an RNA primer
– Leading strand synthesized continuously
– Lagging strand synthesized discontinuously
– Okazaki fragments
– RNA primers are removed and Okazaki fragments
joined by a DNA polymerase and DNA ligase

8. DNA
Figure 8.6

9.  Replication of this DNA molecule always starts at a
certain point (the origin of replication) and it is “semi-
conservative” meaning that one strand in each of the
two resulting double strands is conserved .

10. DNA Replication:
-The identical duplication process of DNA is
termed semi-conservative because the double
strand of DNA is opened up during replication and
each strand serves as the matrix for synthesis of
a complementary strand. Thus each of the two
new double strands “conserves” one old strand.
-The doubling of each DNA molecule begins at a
given starting point called origin of replication.
This process continues throughout the entire
cycle.

12. DNA
• DNA replication is semiconservative
Figure 8.7

13. Translation
• mRNA is translated
in codons (3
nucleotides)
• Translation of mRNA
begins at the start
codon: AUG
• Translation ends at
a STOP codon: UAA,
UAG, UGA
Figure 8.2

14. The Genetic Code
Figure 8.9

15. Structure Of RNA
• Structurally similar to DNA, except for 2
major differences:
– ribose sugar
– uracil in place of thymine.
• 3 types of RNA
– m RNA (messenger RNA)
– t RNA ( transfer RNA )
– r RNA ( ribosomal RNA )

16. Genetic Information In Bacteria
Chromosome Carries properties like virulence,
pathogenicity & resistance
Plasmid Extrachromosomal genetic
material in the cytoplasm
Replicate independently
Bacteriophage Virus infecting bacteria

18. Joshua Lederberg
• The term plasmid was first introduced by Joshua
Lederberg in 1952
• Joshua Lederberg was an American molecular
biologist known for his work in genetics, artificial
intelligence, and space exploration
• He was just 33 years old when he won the 1958
Nobel Prize in Physiology or Medicine for
discovering that bacteria can mate and exchange
genes. He shared the prize with Edward L. Tatum
and George Beadle who won for their work with
genetics

19. What is a Plasmid
• A plasmid is a DNA molecule that is separate
from, and can replicate independently of, the
chromosomal DNA. They are double stranded
and, in many cases, circular. Plasmids usually
occur naturally in bacteria, but are sometimes
found in eukaryotic organisms (e.g., the
2micrometre-ring in Saccharomyces
cerevisiae).

20. Basic understanding of Plasmid
• A plasmid is a vehicle that can carry artificially
inserted DNA. It will replicate in E. coli, and with
its own replication it will also replicate the
inserted DNA, independent of it's origin. In a way
one can see a plasmid as a minute DNA factory.
The main criteria for a 'good' plasmid is that it
takes up the insert you want to put in, and that it
replicates in sufficient amounts,and that it does
not destroy your insert during the process.

21. What is a plasmid?
 A circular piece of
autonomously
replicating
DNA
 Originally evolved by
bacteria
 May express antibiotic
resistance gene
or be modified to express
proteins of interest

22. Bacterial plasmid

23. Plasmid
• Plasmids are molecules of
DNA that are found in
bacteria separate from
the bacterial
chromosome.
• They: are small (a few
thousand base pairs)
usually carry only one or
a few genes are circular
have a single origin of
replication

24. Multiplication of Plasmids
• Plasmids are replicated
by the same machinery
that replicates the
bacterial chromosome.
Some
• plasmids are copied at
about the same rate as
the chromosome, so a
single cell is apt to have
only a single copy of the
plasmid. Other plasmids
are copied at a high rate
and a single cell may

25. Plasmids enters the Bacteria with ease
• Plasmids enter the bacterial
cell with relative ease. This
occurs in nature and may
account for the rapid spread
of antibiotic resistance in
hospitals and elsewhere.
Plasmids can be deliberately
introduced into bacteria in
the laboratory transforming
the cell with the incoming
genes.

26. Understanding a Plasmid
• Many bacteria have accessory DNA
molecules in addition to their larger
chromosome. These molecules,
called lasmids, are extensively used
in genetic engineering. In order to be
useful in labs, these plasmids need to
have an origin of replication (ori),
which enables them to replicate
within a bacterial cell. They also need
to have multiple restriction enzyme
sites to enable cutting and pasting of
DNA into a plasmid.

27. Understanding a Plasmid
• Most plasmids have one or
two identifiable markers
that give a distinct
phenotype to the bacterial
cell. Examples of such
markers include antibiotic
resistance (ampR) or
expression of an enzyme
that catalyzes a reaction
that produces a color
change (lacZ).

28. • Some plasmids have the ampR
gene, which confers resistance to
the antibiotic ampicillin. E. coli
cells containing this plasmid,
termed "+ampR" cells, can survive
and form colonies on LB agar that
has been supplemented with
ampicillin. In contrast, cells lacking
the ampR plasmid, termed "–
ampR" cells, are sensitive to the
antibiotic, which kills them. An
ampicillin-sensitive cell (– ampR)
can be transformed to an
ampicillin-resistant (+ampR) cell by
its uptake of a foreign plasmid
containing the ampR gene
• Plasmid ampicillin
resistance

29. • In microbiology, an extra chromosomal genetic element that
occurs in many bacterial strains. Plasmids are circular
deoxyribonucleic acid (DNA) molecules that replicate
independently of the bacterial chromosome. They are not
essential for the bacterium but may confer a selective
advantage. One class of plasmids, colicinogenic (or Col ) factors,
determines the production of proteins called colicins, which
have antibiotic activity and can kill other bacteria. Another class
of plasmids, R factors, confers upon bacteria resistance to
antibiotics. Some Col factors and R factors can transfer
themselves from one cell to another and thus are capable of
spreading rapidly through a bacterial population. A plasmid that
is attached to the cell membrane or integrated into the bacterial
chromosome is called an episome.
Plasmids and Microbes

30. Plasmids in Genetic Engineering
• Plasmids are extremely valuable
tools in the fields of molecular
biology and genetics, specifically
in the area of genetic
engineering. They play a critical
role in such procedures as gene
cloning, recombinant protein
production (e.g., of human
insulin), and gene therapy
research. In such procedures, a
plasmid is cut at a specific site (or
sites) using enzymes called
restriction endonucleases

31. Structure of Plasmid
• Plasmid size varies from 1 to
over 1,000 kilo base pairs (kbp).
The number of identical
plasmids within a single cell can
range anywhere from one to
even thousands under some
circumstances. Plasmids can be
considered to be part of the
mobilome, since they are often
associated with conjugation, a
mechanism of horizontal gene
transfer

32. Plasmids used as Vectors
Plasmids
•small (1-1000 kb)
•circular
•extrachromosomal DNA
•Growth is independent
of the host’s cell cycle;
amplification of gene
product
•A type of cloning vector
used to carry a gene not
found in the bacterial
host’s chromosome

33. Plasmid
• Plasmid classification is generally based on incompatibility
group (determined by their replication/partitioning functions)
or the genetic information specified by their DNA (Perlin,
2002). Incompatibility grouping had been used to group
plasmid of Pseudomonas species (Jacoby, 1977) and the
Enterobacteriaceae into 26 incompatibility group (Couturier et
al., 1988).
• Most plasmids have a narrow host range allowing only intra-
species transfer and replication. However, a small group of
plasmids called the broad host range (BHR) plasmids (Inc P, Q,
W, N and C) can be transferred and replicated in a wide range
of bacteria (Hill and Top, 1998 ; Dale and Park, 2004). BHR
plasmids may either be self-transmissible (Tra+, Mob+) or
mobilizable but not self-transmissible (Tra-, Mob+) (Perlin ,
2002).

34. PLASMIDS
• Circular DNA molecules
• Important vectors in genetic engineering
• EPISOME
– Plasmid DNA integrated with chromosomal DNA.
• Types of plasmids
– R plasmid (drug resistance): RTF + r determinant
– F plasmid (maleness )

35. Types of plasmids
1. Sex factor plasmids: the cell that possess this plasmid is called F+, male or the
donor cell, while the one that do not possess it is called; F-, or the recipient
cell.
2. R-plasmids (resistance plasmids) responsible for resistance to drug
3. Col- plasmids: responsible for production of bacteriocins.
4. Heavy metal ion resistant plasmids: responsible for resistance to heavy metal
ions that the bacteria may get exposed in the envirnment
5. Plasmids of catabolic activity: responsible for degredation of highly complex
compounds, such as: hydrocarbons
6. Virulence plasmids: reponsible for production of certain virulence factors such
as: toxin , hemolysin, adhesive factors,… etc

36. Mechanisms Of Genetic Variations
• Mutation
• Transfer or exchange of genetic material
1. Transformation
2. Transduction
3. Conjugation
4. Lysogenic conversion
5. Transposition

37. Mechanisms Of Genetic Variations
• Genotype: is the gatalogue of gene arranged on the DNA molecule.
• Phenotype: the collection of characters as result of the expression of
these genes.
• Mutation: hereitiditary changes occur in the genotype which may or may
not lead to phenotypic change.

38. Mutation
• The physical or the chemical agent that leads to mutation is
called mutagen, and the bacteria produced with such
genotypic change are called mutants.
• Mutation occur either spontaneously or by induction.
• Induced mutation occur as a result of the effect of one of the
followings:

39. Mutation
• A-Physical: such as U.V. light, X-ray…etc.
• B-chemical:such as 5-bromouracil, nitrous
acid, hydroxylamine, nitrogen mustard,
• acridines, nitrosoguanidine… etc.
• C-Biological: such as transposons.

40. Mutation
• Types of mutations:
1. Deletion mutation: this result in the loss of a piece of
DNA.
• A B C D E F G______________A B C D F G
•2. Inversion mutation:this result in recombining the
cut piece in revese order.
•A B C D E F G_____________A B C E D F G

41. Mutation
• 3. Insertion mutation: a totally new base sequence is synthesized
and inserted in the DNA molecule.
• A B C D E F G_____________A B C K D E F G
• 4. Substitution mutation: a newly different base sequence is
synthesized instead of the lost one
• A B C D E F G______________A B C N E F G
• 5. Duplication mutation: an insertion of a base sequence similar to a
sequence already existed.
• A B C D E F G______________A B C D B E F G

42. Bacterial genetics
• Experiments by Nature of the genetic material
 Griffith (1928); working on S- forms and R-forms of
the strep. Pneumonia
 Avery (1944); mixing DNA extract of the S-forms
with and without Dnase, then mixing it with R-
forms
 Hershy and Chase (1952), used radioactive isotops
on bacteriophages (S25-for head protein ) and (P32-
for nucleic acids)

43. An "S" or SMOOTH coat strain, which is
lethal to mice.

44. An “R" or rough coat strain, which is
NOT lethal to mice.

45. Griffith found that he could heat inactivate the
smooth strain.

46. heat-inactivated S strain,
mixed with the R strain, the mouse would die.
Thus there was some
Material in the heat-killed S strain that was responsible for
"transforming" the R strain into a lethal form.

47. Gene transfer:
• Recombination ; is the reassortment of nucleotide sequences
within the DNA molecule
• Recombination may occur between
Donor Recepient
Chromosomal
DNA
Plasmid DNA
Viral DNA
Plasmid DNA
Chromosomal DNA
Plasmid DNA
Chromosomal DNA
Chromosomal DNA

48. Sometimes rearrangement occurs within the DNA
molecule itself without an external DNA
Any recombination or spontaneous rearrangement
leads to what is called genotypic change, this may or
may not leads to phenotypic change
The fate of the transferred DNA depends on:
 Its capability to be taken by the host cells
 Its stability within the host chromosome

49. • There are three mechanisms for gene transfer in
bacteria
Conjugation
Transformation Transduction

50. Transformation (Griffith, 1928)
Transfer of genetic information by free DNA. i.e. by direct
uptake of donor DNA by the recipient DNA.
Live noncapsulated (R) pneumococci + heat killed
capsulated (S) pneumococci
Injected into mice
Death of mice
• Live capsulated pneumococcus isolated from the blood
of mice.

52. 3. Transduction
It is the transfer of DNA from a donor to a receptor with
the help of transport bacteriophages.
Bacteriophages
Infection of another bacterium
Transfer of host bacterial DNA to the new bacterium
Acquisition of new characteristics coded by the donor DNA.

53. • Bacteriophage replication occur in 5 stages:
1. Adsorption: where the virus is adsorped on specific receptor on the bacterial
cell wall.
2. Penetration: the virus make a hole in the bacterial cell wall using its core and
its DNA is passed through this hollow core to inside the bacteria
3. Replication : the viral nucleic acid controls the bacterial cell activity and
mechanisms for its benefit and synthesize several numbers of the viral parts.
4. Maturation : during this stage, each head of a virus will be surround one part
of the viral DNA, then the other parts will be joined to each other forming
several numbers of viruses.
5. Release : finally the virus release a lysozyme that leads to cell lysis and the
release of the viruses, then each virus will infect other bacteria, and so on
until the lysis of the whole colony.

55. Bacteriophages
Definition:-
Bacteriophages are viruses that infect bacteria . They are
therefore obligate cell parasites. They possess only one type of
nucleic acid, either DNA or RNA, have no enzymatic systems
for energy supply and are unable to synthesize proteins on
their own.
Morphology:-
Similar to the viruses that infect animals and
vary widely in appearance.

56. Transduction
• Transfer of bacterial
genes via viruses
– Donor to recipient
– Virus: Bacteriophages
• Types
– Generalized
– Specialized
• Replication Cycle
– Lytic
– Lysogenic

57. Composition:
Phages are made up of protein and nucleic acid. The
proteins form the head, tail, and other morphological
elements, the function of which is to protect the phage
genome.
The nucleic acid in most phages is DNA, which occurs
as a double stranded DNA .

58. • Sometimes, the recombination of the viral DNA with the
chromosomal DNA may lead to a drastic change in the
character of the bacteria, such as becoming a powerful toxin
producer (e.g. Clostridium botulinum,, Corynebacterium
diphtheriae… etc).
• The bacteria are called Lysogenic cell, and the
process is called Lysogenic conversion

60. 2. Conjugation
It is the transfer of DNA from a donor to a receptor in a
conjugation process involving cell-to-cell contact.
Conjugation is made possible by two genetic elements:
the conjugative plasmids and the conjugative pilli .
Conjugation is seen frequently in Gram-negative rods
(Enterobacteriaceae), in which the phenomenon has been
most thoroughly researched, and enterococci

62. Transposon (Jumping Genes, Barbara McClintock)
DNA segment that can move
between chromosome & plasmids
Transposons are not self replicative, they depend on chromosomal or
plasmid DNA for replication
Insertion of transposon into a functional
gene would destroy the function of the
gene (internal mutagenic agents)
Plasmid
Chromosome
Transposon

63. • Genetic Engineering – a combination of
methods which allows to conduct artificial
recombination of DNA and produce chimerical
molecules, non-typical for nature

64. • Steps in
1. DNA from any desired source is cleaved into fragments by restriction
endonuclease.
2. The fragments are spliced into vector such as plasmid or viral genome.
3. By transformation, the vector is introduced into a host cell in which it can
replicate.
4. - After replication of the vector & amplification of the original DNA fragment,
vector is isolated.
5. The inserted fragment is cleaved back out & purified.
•see fig.

65. Recombinant DNA

66. • Application of of genetic engineering
(molecular cloning):-
a. Gene structure mapping function e.g. globin.
b. Biosynthesis e.g. insulin, GH (growth hormone), interferon.
c. Control of genetic disease.
d. Using PCR in detection & identification of specific organism e.g. detection of HIV
by PCR.
e. Gene therapy.
•
• NOTE: the field of genetic engineering (molecular cloning) involves the
introduction of new genes into the cells.

67. Chemotherapy
• Refers to treatment of disease by
chemicals that kill cells, specifically
those of micro-organisms or cancer

68. Mechanism of action
Penicillin
Cephalosporins
Vancomycin
Bacitracin
Polymyxin
Colistin
Aminoglycosides
Tetracycline
Chloramphenicol
Macrolide
Lincomycin
Sulfonamides
PAS
INNH
Trimethoprim
Rifampin
Quinolones
Antimetabolites

69. PABA Pteridine
Dihydropteroate synthetase
Dihydropteroic acid
Di hydroFolic Acid
Dihydrofolate reductase
Tetrahydrofolate acid
purines
Nucleic Acid synthesis

70. PABA Pteridine
sulfonamides Dihydropteroate synthetase
Dihydropteroic acid
Di hydroFolic Acid
trimethoprim Dihydrofolate reductase
Tetrahydrofolate acid
purines
Nucleic Acid synthesis

71. Selective toxicity
• An ideal antimicrobial agent
should exhibit ST
• Drug is harmful to parasite without
being harmful to host at the
particular dose
1. Receptor specific
2. Biochemical event

72. Mechanism of Drug Resistance
NON GENETIC AND GENETIC
NON GENETIC-
• Metabolically inactive/non
multiplying
• M.orgs lose specific target site
• Drug unable to penetrate the site of
infection

73. Antimicrobial
Chemotherapy
Modern Chemotherapy-Paul
Ehrlich(18450-1915)-arsenicals for
syphilis and m.blue for malaria
Domagk-1935-Prontosil
Alexander Fleming-Penicillium notatum-
Penicillin
Antibiotic-
Antimicrobial
Chemotherapeutic

74. Drug Resistance
Mutational Transferable
 Decreased permeability to
drug/alt metabolic
path/inactivating enzymes
 Single drug
 Low degree of resistance
 Not transferable
 Metabolically defective
 Virulence maybe lowered
 Combination of drugs useful
 Inactivating enzymes
 Multidrug
 High degree of
resistance
 Transferable
 Metabolically normal
 No decrease in
virulence
 -----------

75. Biochemical mechanism of
Drug resistance
• Production of enzymes that
destroy the active drug
1. Beta lactamases
2. Adenylating/phosphorylating/acetylating
3. Acetyl transferase
• Change of permeability
• Develop altered structural target
• Altered metabolic pathway
• Altered enzyme

76. • References:
• 1- Jawetz, Melnick, & Adelberg’s.( 2013).
Medical Microbiology (Twenty-Sixth Edition).
• 2- Kenneth Todar. (2008).Todar’s Online
Textbook of Bacteriology ,University of
Wisconsin.
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