Microbiology Ch 08 lecture_presentation

PowerPoint® Lecture Presentations prepared by Mindy Miller-Kittrell, North Carolina State University

C H A P T E R

© 2015 Pearson Education, Inc.

Recombinant DNA Technology

8


The Role of Recombinant DNA Technology in Biotechnology• Biotechnology – the use of microorganisms to

make practical products

• Recombinant DNA technology• Intentionally modifying genomes of organisms for

practical purposes

• Three goals

• Eliminate undesirable phenotypic traits

• Combine beneficial traits of two or more organisms

• Create organisms that synthesize products humans need


Figure 8.1 Overview of recombinant DNA technology.Bacterial cell

Bacterialchromosome

Plasmid

Isolate plasmid.

DNA containinggene of interest

Gene of interest

Enzymatically cleaveDNA into fragments.

Isolate fragmentwith the gene ofinterest.

Insert gene into plasmid.

Insert plasmid and gene intobacterium.

Culture bacteria.

Harvest copies ofgene to insert intoplants or animals

Harvest proteinscoded by gene

Eliminateundesirablephenotypictraits

Createbeneficialcombinationof traits

Produce vaccines,antibiotics,hormones, orenzymes

1

2

3

4

5

6


The Role of Recombinant DNA Technology in Biotechnology• Tell Me Why• Why aren't the terms genetic engineering and

biotechnology synonymous?


The Tools of Recombinant DNA Technology

• Mutagens• Physical and chemical agents that produce mutations

• Scientists utilize mutagens to

• Create changes in microbes' genomes to change

phenotypes

• Select for and culture cells with beneficial

characteristics

• Mutated genes alone can be isolated



• The Use of Reverse Transcriptase to

Synthesize cDNA• Isolated from retroviruses

• Uses RNA template to transcribe molecule of cDNA

• Easier to isolate mRNA molecule for desired protein first

• cDNA generated from mRNA of eukaryotes has introns

removed

• Allows prokaryotic cells to produce eukaryotic proteins



• Synthetic Nucleic Acids• Molecules of DNA and RNA produced in cell-free

solutions

• Uses of synthetic nucleic acids

• Elucidating the genetic code

• Creating genes for specific proteins

• Synthesizing DNA and RNA probes to locate specific

sequences of nucleotides

• Synthesizing antisense nucleic acid molecules



• Restriction Enzymes• Bacterial enzymes that cut DNA molecules only at

restriction sites

• Restriction site sequences are usually palindromes

• Categorized into two groups based on type of cut

• Cuts with sticky ends

• Cuts with blunt ends


Figure 8.2 Actions of restriction enzymes.

Restriction site(palindrome)

5′

Restriction enzyme

Sticky ends

Production of sticky ends

Restrictionenzyme 1

Restrictionenzyme 2

Blunt ends

Productionof blunt ends

Restriction fragments from two different organismscut by the same restriction enzyme

Ligase

Recombinant DNA molecules

Recombinants using blunt ends

Ligase

Recombinants using sticky endsRecombinant DNA molecules

3′G A A T T CC T T A A G

5′ 3′C C C G G GG G G C C C

5′ 3′C C C G G GG G G C C C

5′ 3′G T T A A CC A A T T G

5′ 3′G T T A A CC A A T T G

5′ 3′C C C A A CG G G T T G

5′ 3′G T T G G GC A A C C C

5′ 3′A A G C T TT T C G A A

5′ 3′A A G C T TT T C G A A

AT T C G A

A G C T TA A

T T C G A

GC T T A A

A A T T CG

A G C T TA

+



Recombinant DNA Technology



• Vectors• Nucleic acid molecules that deliver a gene into a cell

• Useful properties

• Small enough to manipulate in a lab

• Survive inside cells

• Contain recognizable genetic marker

• Ensure genetic expression of gene

• Include viral genomes, transposons, and plasmids


Figure 8.3 An example of the process for producing a recombinant vector.



• Gene Libraries• A collection of bacterial or phage clones

• Each clone in library often contains one gene of an

organism's genome

• Library may contain all genes of a single chromosome

• Library may contain set of cDNA complementary to

mRNA


Figure 8.4 Production of a gene library.

Genome

Isolate genomeof organism.

Generate fragments usingrestriction enzymes.

Insert each fragmentinto a vector.

Introduce vectorsinto cells.

Culture recombinant cells;descendants are clones.

1

2

3

4

5

1 2 3 4 5 6 7 8 9 10 11

1 2 3

4 5 6

7 8 9

10 11

1 2 3 4 5 6

7 8 9 10 11

1 2 3 4 5 6

7 8 9 10 11



• Tell Me Why• Why did the discovery of restriction enzymes speed up

the study of recombinant DNA technology?


Techniques of Recombinant DNA Technology

• Multiplying DNA in vitro: The Polymerase

Chain Reaction (PCR)• Large number of identical molecules of DNA are

produced in vitro

• Critical to amplify DNA in variety of situations

• Epidemiologists use to amplify genome of unknown

pathogen

• Amplified DNA from Bacillus anthracis spores in 2001 to

identify source of spores



• Multiplying DNA in vitro: The Polymerase

Chain Reaction (PCR)• Repetitive process consisting of three steps

• Denaturation

• Priming

• Extension

• Can be automated using a thermocycler


Polymerase Chain Reaction (PCR): Overview


PCR: Components


Figure 8.5a The use of the polymerase chain reaction (PCR) to replicate DNA.

Denaturation

Priming

Extension

Original DNAmolecule

DNA primerDeoxyribonucleotidetriphosphates

DNA polymerase

Heat to 94°C

Cool to 65°CDNA polymerase

DNA primer

72°C

1

2

3

3′5′

3′

5′

3′5′

3′5′

5′

5′

Repeat4


Figure 8.5b The use of the polymerase chain reaction (PCR) to replicate DNA.


PCR: The Process



• Selecting a Clone of Recombinant Cells• Must find clone containing DNA of interest

• Probes are used



• Separating DNA Molecules: Gel Electrophoresis and the Southern Blot• Gel electrophoresis

• Separates molecules based on electrical charge, size, and shape

• Allows scientists to isolate DNA of interest• Negatively charged DNA drawn toward positive electrode• Agarose makes up gel; acts as molecular sieve• Smaller fragments migrate faster and farther than larger

ones• Determine size by comparing distance migrated to

standards


Figure 8.6 Gel electrophoresis.

Electrophoresis chamber filled with buffer solution

Lane of DNA fragments of known sizes (kilobase pairs)

Agarose gel

DNA

Wire

Wells

Movement of DNA

AB

CD

E

a

b

(50)(40)

(35)

(15)(10)(5)

(+)

(–)



• Separating DNA Molecules: Gel Electrophoresis and the Southern Blot• Southern blot

• DNA is transferred from gel to nitrocellulose membrane

• Probes are used to localize DNA sequence of interest

• Northern blot – similar technique used to detect RNA

• Uses of Southern blots

• Genetic "fingerprinting"

• Diagnosing infectious disease

• Demonstrating presence of organisms that cannot be cultured


Figure 8.7 The Southern blot technique.



• DNA Microarrays

• Consist of molecules of immobilized single-stranded DNA

• Fluorescently labeled DNA washed over array will adhere

only at locations where there are complementary DNA

sequences

• Variety of scientific uses of DNA microarrays

• Monitoring gene expression

• Diagnosing infection

• Identifying organisms in an environmental sample


Figure 8.8 DNA microarray.



• Inserting DNA into Cells• Goal of DNA technology is insertion of DNA into cell

• Natural methods • Transformation

• Transduction

• Conjugation

• Artificial methods• Electroporation

• Protoplast fusion

• Injection – gene gun and microinjection


Figure 8.9a-b Artificial methods of inserting DNA into cells.

ChromosomePores in wall and membrane

Electrical field applied

ElectroporationCompetent cell

DNA fromanother source

Cell synthesizesnew wall

Recombinant cell

Cell walls

Enzymes removecell walls

Protoplasts

Protoplast fusion

Polyethyleneglycol

Fused protoplasts

Recombinant cell

Cell synthesizesnew wall

New wall


Figure 8.9c-d Artificial methods of inserting DNA into cells.

Blank .22-caliber shell

Nylonprojectile

Vent Plate to stopnylon projectile

DNA-coated beads Target cell

Gene gunNylonprojectile

Micropipette containing DNA

Target cell’s nucleus

Target cell

Suction tubeto hold targetcell in place

Microinjection



• Tell Me Why• Why wasn't polymerase chain reaction (PCR) practical

before the discovery of hyperthermophilic bacteria?


Applications of Recombinant DNA Technology

• Genetic Mapping• Locating genes on a nucleic acid molecule

• Provides useful facts concerning metabolism, growth

characteristics, and relatedness to others



• Genetic Mapping• Locating genes

• Until 1970, genes were identified by labor-intensive

methods

• Simpler and universal methods are now available

• Restriction fragmentation

• Determine relative location of DNA fragments

produced by cleavage with restriction enzymes

• Fluorescent in situ hybridization (FISH)

• Fluorescent probe used to visualize location of a gene


Figure 8.10 Fluorescent in situ hybridization (FISH).



• Genetic Mapping• Nucleotide sequencing

• Genomics – sequencing and analysis of the nucleotide

bases of genomes

• Elucidation of the genomes of pathogens is a priority

• Used to relate DNA sequence to protein function


Figure 8.11 Automated DNA sequencing.




• Environmental Studies• Most microorganisms have never been grown in a

laboratory

• Scientists know them only by their DNA fingerprints

• Allowed identification of over 500 species of bacteria from

human mouths

• Determined that methane-producing archaea are a

problem in rice agriculture



• Pharmaceutical and Therapeutic Applications• Protein synthesis

• Creation of synthetic proteins by bacteria and yeast cells

• Vaccines

• Production of safer vaccines

• Subunit vaccines

• New approaches to stimulate immunological memory

• Introducing genes of pathogens into fruits and vegetables

• Injecting humans with plasmid carrying gene from pathogen

• Humans synthesize pathogen's proteins



• Pharmaceutical and Therapeutic Applications• Genetic screening

• DNA microarrays are used to screen individuals for

inherited disease caused by mutations

• Can also identify pathogen's DNA in blood or tissues

• DNA fingerprinting

• Identifying individuals or organisms by their unique DNA

sequence


Figure 8.12 DNA fingerprinting.



• Pharmaceutical and Therapeutic Applications• Gene therapy

• Missing or defective genes are replaced with normal copies

• Some patients' immune systems react negatively

• Medical diagnosis

• Patient specimens can be examined for presence of gene

sequences unique to certain pathogens

• Xenotransplants

• Animal cells, tissues, or organs introduced into human body



• Agricultural Applications• Production of transgenic organisms

• Recombinant plants and animals altered by addition of

genes from other organisms



• Agricultural Applications• Herbicide tolerance

• Gene from Agrobacterium tumefaciens conveys

resistance to glyphosate (Roundup)

• Farmers can kill weeds without killing crops

• Salt tolerance

• Scientists have inserted a gene for salt tolerance into

tomato and canola plants

• Transgenic plants survive, produce fruit, and remove salt

from soil



• Agricultural Applications• Freeze resistance

• Crops sprayed with genetically modified bacteria can tolerate

mild freezes

• Pest resistance

• Bt toxin

• Naturally occurring toxin harmful only to insects

• Used by organic farmers to reduce insect damage to crops

• Gene for Bt toxin is inserted into various crop plants

• Genes for Phytophthora resistance are inserted into potato

crops


Figure 8.13 Genetically modified papaya plants.



• Agricultural Applications• Improvements in nutritional value and yield

• Enzyme that breaks down pectin is suppressed in some

tomatoes

• Allows tomatoes to ripen on vine and increases shelf life

• BGH allows cattle to gain weight more rapidly

• Have meat with lower fat content and produce 10% more

milk

• Gene for β-carotene (vitamin A precursor) are inserted into rice

• Scientists considering transplanting genes coding for entire

metabolic pathways



• Tell Me Why• Why don't doctors routinely insert genes into their

patients to cure the common cold, flu, or tuberculosis?


The Ethics and Safety of Recombinant DNA Technology• Long-term effects of transgenic manipulations are

unknown

• Unforeseen problems arise from every new technology and procedure

• Natural genetic transfer could deliver genes from transgenic plants and animals into other organisms

• Transgenic organisms could trigger allergies or cause harmless organisms to become pathogenic


The Ethics and Safety of Recombinant DNA Technology• Studies have not shown any risks to human health

or environment

• Standards are imposed on labs involved in

recombinant DNA technology

• Can create biological weapons using same

technology


The Ethics and Safety of Recombinant DNA Technology• Ethical issues

• Routine screenings?

• Who should pay?

• Genetic privacy rights?

• Profits from genetically altered organisms?

• Required genetic screening?

• Forced correction of "genetic abnormalities"?


The Ethics and Safety of Recombinant DNA Technology• Tell Me Why• Why don't scientists who work with recombinant DNA

know all the long-term effects of their work?