Transcript Presentation 3

Genetically Engineered Microbes
GEM
Wilfred Mbacham, MS, DS, MPH, ScD
Professor of Public Health Biotechnology
Fellow of the Cameroon Academy of Science
Department of Biochemistry, Physiology and Pharmacology
University of Yaounde I
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Where Hence Has Thou Come?
Microbes
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Virtually every environmental niche
Extremes of pH and salinity
Extremes of temperature and pressure
Without air (Anaerobic)
Growth on many chemical substrates
Attached to surfaces in biofilms
Geothermal vents and subterranean deposit
History of GEM
S 1953 - Discovery of DNA Structure
S 1973 - First recombinant bacteria i.e., Escherichia .coli
expressing a Salmonella gene.
S 1978 - E. coli strain producing the human protein insulin
by Herbert Boyer’s Company, Genentech,
S 1986, field tests of bacteria genetically engineered to
protect plants from frost damage (ice-minus bacteria) Advanced Genetic Sciences of Oakland, California,
Transgenes are built from
Modified Microbial Plasmids
The Diversity is Enormous
16s rRNA sequences reveal
true diversity in soil DNA
Requirement for Biodegradation
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Nutrients
Carbon, Nitrogen, Phosphorus, Sulfur
Many chemicals supply these
Micronutrients/ trace metals/ vitamins
Electron acceptors - usually O2
Converts / burns carbon substrate to CO2
Energy and biomass ie GROWTH
Genetically Engineered Microbes:
Wide Application
GMOs have widespread applications. Genetically modified
microbes can be used for the following applications:
1. Bioremediation – Oil Spills
2. Industry - Enzyme
3. Agriculture - Pesticide
4. Medicine - Insulin
5. Energy Production - Methane
Bioremediation starts with an
Aerobic Biodegradation
O2 consumption
GROWTH - CELL DIVISION
INCREASE IN BIOMASS
2.0m
ORGANIC POLLUTANT
AND NUTRIENTS
(C,P,N,O,Fe,S……)
SINGLE
BACTERIUM
Controlled release of energy
Slow Burning!
CO2 evolved
Oxygen and Electron Acceptors
in Biodegradation
2H+
Electron acceptor
O2
SUBSTRATE
ADP
METABOLISM
CARBON
Pi
ENERGY
GROWTH/Biomass
CO2
ATP
H2/2e-
H2 O
Fixation of Oxygen is the first
Step in Biodegradation
Cell membrane
NAD+
ReductaseNAP
(OX)
FerredoxinNAP
(OX)
ISPNAP
(OX)
O2
OH
OH
NADH
+ H+
ReductaseNAP
(RED)
FerredoxinNAP
(RED)
Cell Biomass
CO2
ISPNAP
(RED)
Further degradation
Anaerobic Growth and Biodegradation
Organic matter
Fermented
Acetic Acid
+
Methanogenesis
CH4 CO2 H2O
H2 CO2
GEM Biodegradation of
PolyAromatic Hydrocarbons
S PAHs - Naphthalene, Phenanthrene, and Anthracene, whose
occurrence in the soil is due to spills or leakage of fossil fuels or
petroleum products.
S Pseudomonas fluorescens isolated from PAH contaminated soils, was
genetically engineered with lux genes from Vibrio fischeri, a
bacterium that lives in the light generating organisms of certain
deep sea fish.
S The modified strain, P. fluorescens HK44 responds to naphthalene
by luminescence, which can be detected with the help of light
sensing probes.
S This will allow the detection of PAHs in the contaminated soils for
Biodegradation
GEM for Treating Oil Spills
Dr. Ananda Mohan Chakrabarty, USA
S The first genetically engineered organism for
bioremediation Pseudomonas,
S Capable of degrading 2,4,5-trichlorophenoxyacetic acid
(2,4,5-T).
S The strain contained two plasmids, each providing a
separate hydrogen degradative pathway,
Heavy Metal Sequestration by
GEM
S GEM sequesters Heavy Metal in the soil and makes it non-
available to Plants and Animals
S Sequestration of Cadmium was achieved by transfer of a mouse
gene, encoding metallothionein to a Ralstonia eutropha (a natural
inhabitant of soil).
S Metallothionein in this GEM was expressed on the outer surface
of the cells to help in sequestering of cadmium.
S Steps – 1. Synthesis and export of MTβ – 2. Genetically
engineered Ralstonia eutropha soil – 3. Innoculation - Cdsensitive plant can grow
Unresolved Issues in
Bioremediation
Many issues remain to be resolved before this method is
adopted widely. Priority areas of research include the
following:
 Improving microbial strains;
 Improving bioanalytical methods for measuring the level of
contaminants
 Developing analytical techniques for better understanding,
control and optimization of environmental and reactor
systems
GEM in Bioterrorism - I
S Genetically engineered bioweapons, detection or
vaccines are easily sidestepped by the artificial
microbes.
S Strengthen the Bioweapons Convention and establish a
verification system.
GEM in Bioterrorism - II
S Example 1: Bacteria causing unusual symptoms
S Francisella tularensis, the causative agent of tularemia and a well
known biological weapon agent made the bacteria produce betaendorphin, an endogenous human drug, which caused changes
in the behaviour of mice when infected with the transgenic
bacteria.
S Example 2: Transferring a lethal factor to harmless human gut
bacteria - lethal factor of Bacillus anthracis, the causative agent of
anthrax, and introduced into Escherichia coli, a normally
harmless gut bacteria.
GEM in Bioterrorism - III
S Example 3: Antibiotic resistant anthrax and tularemia. An antibiotic
resistance marker gene (tetracyclin) was been inserted into Francisella
tularensis sub sp. holarctica bacteria (5), a close relative of the causative agent of
tularaemia. these bacteria making agents less treatable.
S Example 4: Invisible anthrax - In December 1997, the same Russian research
group from Obolensk by putting new genes into fully pathogenic strains of
anthrax, the scientists altered anthrax’s immunopathogenic properties,
making existing anthrax vaccines ineffective against the new geneticallyengineered types.
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GEM in Industry
S Commercial production of some non microbial products such as
insulin, interferon, human growth hormone and viral
vaccines.
S Energy sectors - Production of single cell proteins (SCP) to meet
food and fodder problems, and for biogas production to provide
energy to electrify villages.
S Microbes are also being used to meet effectively the crisis in both
environment and energy sectors. Recovery of metals from polluted
waterwaysElimination of sulphur from metal ores and coal fired power and
ii. Use of biofertilizers and biopesticides
i.
Industrial Products of GEM
S Various foods and drinks
S Enzymes for varied uses (GM enzymes); biocatalysts
S Engineered proteins ( antibodies )
S Vaccines and antibiotics (secondary metabolites)
S Primary metabolites and bulk chemicals (amino acids (glutamic
acid) and organic acids (acetic acid)
S Pharmaceuticals and novel chiral chemicals
S Recovery of metals in bioleaching
S Biosensors (use of enzymes to specifically detect chemicals in
medical and )
Biofermenters for Industrial Purposes
GEM in Agriculture
Different application of GMO in production of crops which
resist different types of viral, bacterial and insect pest :
Potato - modified to produce a beetle killing toxin
Yellow squash – modified to contain viral genes that
resist to the most common viral diseases
Develop foods that contain vaccines and antibodies that
offer valuable protection against diseases such as cholera,
hepatitis, and malaria
Canola – modified to resist one type of herbicide or
pesticide
GMO Bans and Acceptances
Great Concern with GEM
Following issues are of great concern regarding GMO
1. Fundamental weaknesses of the concept
2. Health hazard and environmental hazard and
related food safety
3. Increased corporate control of agriculture and
unintended economic consequences
Fundamental Weaknesses
S Imprecise Technology
S A gene can be cut precisely from the DNA of an organism, but the insertion into
the DNA of the target organism is basically random. As a consequence, there is a
risk that it may disrupt the functioning of other genes essential to the life of that
organism. (Bergelson 1998)
S Side Effects
S Genetic engineering is like performing heart surgery with a shovel. Scientists do
not yet understand living systems completely enough to perform DNA surgery
without creating mutations which could be harmful to the environment and our
health. They are experimenting with very delicate, yet powerful forces of nature,
without full knowledge of the repercussions. (Washington Times 1997, The Village
Voice 1998)
Fundamental Weaknesses
S Widespread Crop Failure—
S Genetic engineers intend to profit by patenting genetically engineered seeds. This
means that, when a farmer plants genetically engineered seeds, all the seeds have
identical genetic structure. As a result, if a fungus, a virus, or a pest develops
which can attack this particular crop, there could be widespread crop failure.
(Robinson 1996)
S Threatens Our Entire Food Supply—
S Insects, birds, and wind can carry genetically altered seeds into neighboring fields
and beyond. Pollen from transgenic plants can cross-pollinate with genetically
natural crops and wild relatives. All crops, organic and non-organic, are vulnerable
to contamination from cross-pollinatation. (Emberlin et al 1999)
GMO Activities
Four Risk Categories
S Category I
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comprises routine recombinant DNA experiments conducted
inside a laboratory;
S Category II
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consists of both laboratory and greenhouse experiments
involving transgenes that combat biotic stresses through
resistance to herbicides and pesticides;
S Categories III and IV
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comprise experiments and field trials where the escape of
transgenic traits into the open environment could cause
significant alterations in the ecosystem
Cameroon Needs
 To regulate GMOs under the Biosafety Law
 Rules for the “Manufacture, Use, Import, Export and
Storage of Hazardous Microorganisms, genetically
Modified Organisms and Cells”
 Safety guidelines on the Use of Recombinant DNA
 Guidelines for management of Toxicity and Allergenicity
of Transgenic organisms
Remember what a GEM is?
S A genetically Engineered Microbe (GMO) is one
whose genetic material has been altered using
recombinant DNA technology from different
sources making them transgenic. Transgenic
organisms, a subset of GMOs, are organisms
which have inserted DNA that originated in a
different species.
Acknowledgement
SC Santra - GMO and GMO Controversies. Dept. of
Environmental Science, University of Kalyani,
Kalyani, Nadia, [email protected]
MJ Larkin - Microorganisms in the Environment and
Microbial Biotechnology,