M. genitalium

igocheddarBiotechnology

Dec 14, 2012 (4 years and 8 months ago)

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Biotechnology

Creating new pharmaceuticals

Creating new diagnostic tools

Creating new crops/foods

Cleaning up the environment


Genetic Engineering


transferring
natural or synthetic genes to an organism


Recombinant DNA


combining DNA
from two or more organisms

http://www.cyberartsweb.org/cpace/cyborg/cyborgfood/images/gmo_tomato_b2.jpg

Fig. 9.1

Recombinant DNA


Laboratory procedures require the
services of microorganisms:


Enzymes for cutting and pasting DNA


Replication of DNA sequences


Production of recombinant proteins

How to make Recombinant
DNA


It’s all too easy


DNA can be isolated from almost any
living cell in a matter of a couple of hours


Once isolated, restriction enzymes are
used to cut the DNA into fragments


The fragments can be mixed and
matched


DNA ligase seals them together

Restriction Enzymes


Cut DNA at specific sequences


Many leave “sticky ends”


a single
-
stranded overhang


Matching sticky ends from different DNA
molecules can form base pairs with each
other


DNA ligase seals up the paired sticky
ends

Fig. 9.2

Plasmids


Recombinant DNA molecules are often
put into plasmids


Plasmids enable the DNA to be
replicated by bacterial cells, usually
E.coli

Recombinant Pharmaceuticals


This same concept has been used to
make:


Human insulin


Human growth hormone


Bovine growth hormone


Human clotting factors

Recombinant Gamma Interferon
in
E. coli

Fig. 9.13

Table 9.1

“Visualizing” DNA


The details of a DNA molecule (its
sequence) cannot be observed directly


The size of a DNA molecule can be
determined


Gel electrophoresis is a way of separating
differently sized DNA molecules

Gel Electrophoresis

An electric current is able to stimulate the migration of DNA

through the gel matrix since DNA has a negative charge.

Transforming Plants


A “gene gun” is used


DNA is coated onto gold particles, which
can penetrate the plant cell wall


A small percentage of cells will take up
the foreign DNA


Again, a selection process makes things
easier

Fig. 9.6

See also fig. 9.20

Transforming Animals


DNA can be microinjected into the
nucleus


Recombination is required for the DNA to
be stably incorporated


Often done with eggs or embryos

Fig. 9.7


GE = genetically engineered


GMO = genetically modified organism


Transgenic = genetically engineered


Transgene = one that has been
engineered into an organism

GE Food


Corn, soy, and other plants have an
herbicide
-
resistance gene from
Salmonella



“Round
-
Up Ready” from Monsanto


Corn, potato, and other plants have an
insecticide gene from
Bacillus
thuringiensis



Bt
-
corn, etc. from Monsanto

More GE Food


Corn has been genetically engineered to
produce antibiotics


animal feed.


Rice has been genetically engineered to
produce
b
-
carotene


“Golden Rice.”


Salmon have been genetically
engineered with extra growth hormone
genes.


Many more.

Genetic Engineering


Why does genetic engineering work (at
least sometimes)?


What are the limits to predicting an
outcome with genetic engineering?



The chemical structure of DNA, RNA, and
proteins


same in all life


Gene expression follows same pattern in
all life


Genetic code is nearly universal

BUT


Environmental effects on gene
expression


Other unpredictable factors

Concerns with GMOs


Safe to eat? Production of allergens?


Round
-
Up Ready crops actually encourage greater
use of chemicals


Bt
-
crops contain pesticide


Short
-
term testing (often conducted by the
company)


Environmental contamination


pollination of wild
relatives (big concern for Pharma
-
crops)


Ecological effects


the pollen can end up in
unexpected places (pollen cannot be contained)


Should food crops (or any biological material) be
patented?

Labeling of Food


No requirement for GMOs to be labeled
as such


“GMO
-
Free” labeling


Monsanto has threatened to sue


Environmental Contamination


With pollen from faster
growing varieties


With pollen from “heartier”
varieties


With pollen from
experimental “terminator”
varieties



Scientific Study


Evidence for transgenic corn
contamination has been found in wild
maize in Chiapas, Mexico. [Quist &
Chapela (2001)
Nature 414
: 541
-
3.]


Heated Controversy


Nature
retracted the article after criticism
from pro
-
Biotech scientists


Chapela’s tenure fight at UC Berkeley


Committee votes him in


Administration says no


Reversed two years later (early ’05)

Industry Money in Academia


In 1998, Novartis (now Syngenta) & UC
Berkeley begin a partnership


Novartis gives $25M over 5 years


Novartis gets patent rights for any new
discoveries


Affects on basic research?

GE
-
Free Zones


Several local elections have had
propositions


Many have passed


To be challenged in the courts or through
legislation

Mexico


Has had a ban on GE
-
corn


Other countries


EU, Japan, Brazil

Refusal of Aid


In 2002, Zambia refused GE corn (unless
it was ground into meal)


Offered by U.S. during Zambia’s drought

Concerns with GMOs


Safe to eat? Production of allergens?


Round
-
Up Ready crops actually encourage greater
use of chemicals


Bt
-
crops contain pesticide


Short
-
term testing (often conducted by the
company)


Environmental contamination


pollination of wild
relatives (big concern for Pharma
-
crops)


Ecological effects


the pollen can end up in
unexpected places (pollen cannot be contained)


Should food crops (or any biological material) be
patented?



Ecological Effects I


Corn is often grown next
to milkweed plants


Milkweed is the food for
Monarch butterfly
caterpillars


Corn pollen lands on
milkweed plants


Cornell Univ. study
suggests that Bt
-
corn
pollen is toxic to the
caterpillars (1999)

Ecological Effects II



Started planting Bt
-
cotton 3
years ago.


17% reduction in
Actinomycetes.


14% overall reduction in
bacteria.


8.9% reduction in total
microbial biomass.


26.6% reduction in Acid
Phosphatase.


22.6% reduction in
Nitrogenase.

http://www.i
-
sis.org.uk/BtCottonKillsSoilandFarmers.php

Ecological Effects III


Midwestern U.S. stream ecology.


Caddis Fly larvae feed on plant debris.


Food for various fish.


BT
-
corn debris seems to affect their vitality (as
compared to non
-
GMO corn).





Rosi
-
Marshall et al., (2007)
PNAS 104:
16204.

Concerns with GMOs


Safe to eat? Production of allergens?


Round
-
Up Ready crops actually encourage greater
use of chemicals


Bt
-
crops contain pesticide


Short
-
term testing (often conducted by the
company)


Environmental contamination


pollination of wild
relatives (big concern for Pharma
-
crops)


Ecological effects


the pollen can end up in
unexpected places (pollen cannot be contained)


Should food crops (or any biological material) be
patented?


Monsanto Sues Canadian Farmer


Percy Schmeiser is (was) a
Canola farmer


He did not plant Round
-
Up
Ready Canola


(Monsanto’s records show
that he did not purchase
any of their seeds)


Monsanto found evidence
of Round
-
Up Ready
transgenes in his crops


They sued him for patent
infringment

Increased Yields?


A major justification for the need for GE
crops is to increase yields


However, this has not yet been
demonstrated


Are food shortages the problem behind
world hunger?

Natural Selection


What effects will genetic engineering
have on the evolution of these (and
related) species?



Again, keeping a high level of biodiversity
is the key to preparedness for unforeseen
environmental changes

Genetically Engineered Humans?


Yes, they exist!

Gene Therapy


Compensates for a “defective” gene.


Replacement is very tricky.


Introduces a “working” gene.


There are many disorders that have a
genetic basis, i.e. hemophilia, cystic
fibrosis


Viruses are often genetically engineered
to transfer the gene to live cells

Gene Therapy Safety Concerns


Viral DNA can randomly integrate into
host genome


can disrupt important
genes (has led to cancer in some
patients)


Two different viruses can recombine with
each other if they infect the same cell


Immune system may “over
-
react”


2 prominent deaths (Jesse Gelsinger in
1999, Jolee Mohr in 2007)

French Trials of SCID Treatment


Severe Combined Immune Deficiency


Genetic deficiency


lack of single gene
product


Gene therapy showed good success rate


But many patients developed leukemia

Synthetic Biology


Designing organisms from “scratch.”


Choose/create a genome and introduce
it into living cells.


Can be for industrial purposes.


Or as a weapon.


Or to design your child.

Mycoplasma genitalium


Organism with smallest known genome.


May be the “simplest” living cell.



Used in initial experiments (other species
too).


J. Craig Venter Institute.

Mycoplasma mycoides

created at the Venter Institute


by swapping a new genome into
Mycoplasma capricolum


3 Milestones


Transfer genome of
M. mycoides

to
another (related) cell


replaces
recipient’s genome.


Construct genome of
M. genitalium
in
vitro.


Transfer this synthetic genome to a living
cell (
Mycoplasma laboratorium
).

Milestones


First 2 were achieved quickly.



Unknown whether the synthetic genome
will actually support a living cell.


Synthetic DNA may lack the necessary
structure and accessories.



3
rd

was achieved eventually.


A Synthetic Virus


A synthetic polio virus has
been created.






Small pox and H5N1
genomes have been
published!!!

The Future of Synthetic Biology?


Stock
M. genitalium
with enzymes to make
biofuels or other industrial products.


Create a novel biological weapon


a cell that
produces neurotoxin, for example.


Create a novel human genome and transfer it
to a one
-
cell stage embryo.


Venter claims this may be possible within 10 years.



More Biotech


Vaccines, Antimicrobials, Antivirals


Aging


Cancer


Cloning


Stem Cells


still more


obesity, diabetes, depression

Summary of Ethical Issues

(some of them)


How can there be adequate testing of biotech
products?


Health, ecological/environmental


Is their proper justification for the development
of the products in the first place?


Reduction of chemical use? Need for greater food
supplies?


Should it be possible to have ownership of
biological materials?


Patenting a corn plant or a cow?