Download Synthetic Biology: Gene Transformation and Protein Purification

Synthetic Biology:
Genetic Transformation
by Steve Post
Life Technologies / IISME
Summer 2011
Synthetic Biology:
What is Synthetic Biology?
Synthetic Biology
• The design and construction of new biological
functions and systems not found in nature.
• Synthetic Biology is an expansion of
biotechnology, to design and build engineered
biological systems that:
– process information, manipulate chemicals,
fabricate materials and structures, produce
energy, provide food, and maintain and enhance
human health and our environment.
Synthetic Biology:
Which organisms are best to transform?
E. Coli bacteria will
grow billions of
offspring in colonies
overnight
• One of the most commonly transformed
organisms is bacteria (small, simple
chromosomes, grow quickly, divide by
cloning, easy to manipulate)
Synthetic Biology:
What do you want to create with DNA?
Glow in the
dark E. Coli
Bacteria
• Genetic Transformation allows scientists to
modify and create genes with nearly endless
possibilities.
Synthetic Biology:
What is genetic transformation?
Transformation
• The insertion of new DNA into a host cell to give
the cell the ability to produce new proteins and
new traits.
• Transformation can be used for:
– Agriculture: plants become more resistant to pests,
frost, drought, etc
– Bioremediation: bacteria can be transformed to have
genes that enable them to digest oil spills
– Production of medicine: bacteria can be transformed
to produce proteins like insulin to help diabetics.
Synthetic Biology:
What is genetic transformation?
Transformation
Requirements:
• Plasmid Vector
• DNA with Gene
• Recombination
• Insert into
Bacteria Cell
Synthetic Biology:
How do you insert genes?
Vector
• The vector is the method used for inserting
genes into a cell during transformation.
– (Example: Viruses are vectors)
• One of the most simple and common vectors
in synthetic biology is a bacterial plasmid.
• Plasmids are small, simple rings of DNA that
can be easily manipulated.
Synthetic Biology:
How do you use a Plasmid as a Vector?
• Bacteria cells have a chromosome and one or more
small circular rings of DNA called plasmids.
• Plasmids can be replicated and pass from one bacteria
to another. This occurs naturally to help bacteria adapt
to new environments.
• Scientists can simulate this process by opening the
bacteria cell membrane and insert a genetically
engineered plasmid as a vector.
Transformation with Plasmid Vector
How do you use a Plasmid as a Vector?
Bacteria cells divide by
cloning themselves, so all
their billions of offspring
have the identical
transformed genes.
Synthetic Biology:
So what traits should we give our bacteria?
• Why not a green glow trait?
• Green Fluorescent Protein
glows bright green.
• Fluorescence is created
when a substance absorbs
one wavelength of light and
emits a lower wavelength.
Synthetic Biology:
What is Green Fluorescent Protein?
Green Fluorescent Protein (GFP)
• Green fluorescent protein (GFP) is a protein
composed of 238 amino acids that exhibits bright
green fluorescence when exposed to UV light.
• GFP first isolated from a jellyfish
• GFP is used a marker gene : GFP is grouped with
other genes in transformation, and the presence of
green fluorescence from a cell is used as an
indicator of successful transformation.
Synthetic Biology:
Where did GFP come from?
• GFP was first isolated from the jellyfish Aequorea
victoria that glows in dark environments.
• The gene that codes for GFP is often used in
genetic engineering as a visible sign of success.
Synthetic Biology:
What can you do with GFP?
Normal Light
UV Light
Colony #3
Colony #3
Colony #2
Colony #2
Colony #1
Colony #1
No Glow!
• GFP is used an success indicator for desired genes.
• Bacteria colonies that glow green under UV light show
successful transformation.
• Colonies that do not glow show errors or mutations.
Synthetic Biology:
What can you do with GFP?
Normal Light
UV Light
Colony #3
Colony #3
Colony #2
Colony #2
Colony #1
Colony #1
No Glow!
• Glowing colonies can be isolated and cloned
to make sure every bacteria cell has the exact
genes that the scientist wants.
Synthetic Biology:
What can you do with GFP?
• Mutations of GFP create a variety of colors
• Bacteria art using the mutant GFP color pallet.
Synthetic Biology:
What can you do with GFP?
“Glo Fish” GFP Zebra Fish
Normal Zebra Fish
• Bacteria are not the only GFP species…
• “Glo Fish” are Zebra fish engineered to be fluorescent.
• Glo Fish were outlawed in California in 2004.
Synthetic Biology:
How do you transform a multi-cellular organism?
• To transform a multi-cellular organism,
scientists manipulate the gametes or zygote.
• Cell division copies the genes during
development, so every cell in the organism
has the modified genes.
Synthetic Biology:
What else can you do with GFP?
Fluorescent Bunnies??
Synthetic Biology:
What can you do with GFP?
•Fluorescent Bunnies = YES!
•Eduardo Kac claims he made
a GFP rabbit as artwork. In
fact, many rabbit embryos
were transformed by
scientists.
•The famous glowing rabbit’s
name was Alba, she was an
albino rabbit that glows
under UV light.
Synthetic Biology:
What can you do with GFP?
•Many species have been transformed with GFP, here
are more realistic photos of GFP mice:
Synthetic Biology:
Is there anything that shouldn’t glow green?
Synthetic Biology:
Is there anything that shouldn’t glow green?