Transcript What`s the Big Idea? Using Bacteria to make proteins

WHAT’S THE BIG IDEA? USING
BACTERIA TO MAKE PROTEINS
Dr. Patricia (Trish) Willy
SCBC Science Outreach Coordinator
Why Should Students Study Biotech?
• ABE mimics the research and development
process used for the recombinant products that
are currently available to treat a wide range of
diseases.
• For example: insulin, epogen, neupagen
• The life sciences/biotech sector is a high growth,
high wage industry for employment in San Diego
• Scientifically literate citizens
www.amgenbiotechexperience.com
Scientific Contribution
Problem-Illness: How can proteins
(drugs/pharmaceuticals) be mass produced?
Solution-Recombinant DNA Technology:
Identify and take the gene for the protein
from the organism that makes it and put it
into another organism that will make large
quantities of that protein.
www.amgenbiotechexperience.com
How do Scientists do it?
Making a Protein-rfp (i.e.,insulin):
Process
– Find the gene that makes the protein
– Cut the gene out (with Restriction Enzymes)
– Put the gene into a carrier (Plasmid/Vector)
– Use the carrier to insert the gene into bacteria (Recombinant
Plasmid with rfp gene)
– Turn on the gene (with arabinose) so the bacteria makes the
protein (red fluorescent protein)
www.amgenbiotechexperience.com
What is diabetes?
• Physiological condition in which the cells of
the body are not able to obtain glucose
circulating in the blood
• Caused by an endocrine disorder in which the
body does not properly use or produce
sufficient quantities of the hormone known as
insulin
• Hyperglycemia is one of first symptoms –
requires constant monitoring of blood-glucose
levels
www.amgenbiotechexperience.com
How your body uses glucose
• The process of digestion chemically breaks down starch
(complex carbohydrate) into the simple sugar known as
glucose
• Glucose is next absorbed by cells of the gastrointestinal
tract and then enters into the bloodstream where it is
distributed to all cells of the body and used as fuel
www.amgenbiotechexperience.com
How your body uses glucose (cont’d.)
• When glucose enters cells, it is metabolized
(enzymatically broken down) in the presence of
oxygen to make ATP (while releasing carbon
dioxide and water)
• ATP = energy currency of the cell – used to
perform work that keeps cells alive
C6H12O6 + O2
glucose
(from food)
balance)
oxygen
(in air)
ATP
adenosine
triphosphate
(energy)
www.amgenbiotechexperience.com
+
CO2
+
carbon
dioxide
(exhaled waste)
H2O
water
(fluid
The importance of insulin
• Chemical messenger (hormone) produced by the
pancreas (beta cells)
• Binds to receptors on the surface of cells
triggering a signal pathway that results in the
aggregation of glucose-transport proteins (GLUT
4) that create channels through which glucose
can diffuse from blood into cells
Summary: Without insulin/receptor
binding, glucose cannot get from blood
into cells resulting in energy deprivation
and death
www.amgenbiotechexperience.com
Type 1 diabetes
• Formerly known as insulin-dependent diabetes
• Results from destruction of pancreatic beta cells
(due to autoimmunity)
• Insulin production of the pancreas is little to
none
• Most common among individuals from northern
European descent
• Onset usually before the age of 20
• Treated with insulin injections
www.amgenbiotechexperience.com
Type 2 diabetes
• Formerly known as non-insulin-dependent
diabetes
• Results from 1) decreased insulin production or
2) desensitization of target cells to insulin
• Many type 2 diabetics have normal insulin levels
• Most common among obese individuals over the
age of 35
• Constitutes more than 90% of all diabetes cases
• Controlled through diet, weight loss, medications
that lower blood glucose, or insulin injection
www.amgenbiotechexperience.com
Sources of insulin – in the beginning
• For 60 years (1922-1982) the only sources of
insulin for use by diabetics were porcine (pig)
and bovine (cow) – virtually identical to human
insulin
• Initially, impurities in animal-derived insulin
resulted in side effects (allergic reactions)
• With increased quality/purification, supply &
demand became a concern in later years
• Thus, scientists began searching for new ways to
synthesize human insulin efficiently in large
quantities
www.amgenbiotechexperience.com
Sources of insulin – a new era
• In 1982, scientists genetically engineered E. coli
bacteria to produce human insulin
• Using recombinant DNA technology, the DNA
sequence coding for human insulin protein was
isolated and inserted into bacterial plasmid
• The recombinant DNA was then introduced into
bacterial host cells
• Genetically modified microbes are currently
primary source for commercially-synthesized
human insulin produced in mass quantities
www.amgenbiotechexperience.com
Making insulin in bacteria: a summary
www.amgenbiotechexperience.com
A success story
• Although not a cure, recombinant human
insulin serves as an excellent example of the
utility and promise of genetic engineering as it
is practically applied in the field of medicine
• Within the last four decades, genetic
engineering has helped to advance
agricultural, medical, and environmental
sciences
• increased food production
• treatments for disorders/diseases
• bioremediation
www.amgenbiotechexperience.com
Using insulin as a model
• The steps in recombinant insulin production
serve as a template regarding the
methodologies used in the Amgen lab series
restriction
enzyme
Plasmid
Purified
Protein
Product
foreign gene
+ ligase
Recombinant plasmid
Digested plasmid
lyse cells; separate product
from bacterial
proteins
recombinant plasmid
Introduced into host
cell
divides
“Transformed” E. coli cell
Transcription & translation yields protein
product from gene insert
www.amgenbiotechexperience.com
Additional Resources
• http://www.dnaftb.org/34/animation.html
• https://www.amgenbiotechexperience.com/si
tes/abe.edc.org/files/abe_english_student_all
_sequences_05.15.15.pdf (pgs. A-7 through A12)
www.amgenbiotechexperience.com