Download Biotechnology part 1

Biotechnology
Biotechnology: The use of
microorganisms, cells or cell components
to make a product.
Genetic Engineering: inserting genes into
cells for biotechnological purposes.
Introduction
• Today’s lecture will focus on Fig. 10.9 on page
297.
• We will talk about how we get from isolating a
desired gene product to the actual production of
the protein or enzyme.
• Realize though, that the techniques we talk about
as well as the others mentioned in your textbook
are only a few of the tools available for research
and genetic engineering, and others are being
developed all the time.
Overview of Genetic Engineering
• Let’s say we want to isolate the gene for human insulin
and put it into E.coli so it can make insulin. The following
are the basic steps required to do that.
• 1. Select a vector (plasmid) for our desired gene.
– A vector is a piece of DNA that acts like a taxi for a selected gene.
• 2. Original DNA cleaved by restriction enzymes to isolate
the desired gene.
• 3. The chosen vector is cleaved by the same restriction
enzymes and the selected gene is inserted.
• 4. The vector is transformed into the target cell.
• 5. Test the microbe for presence of the new gene.
• 6. The engineered microbe is grown and gene copies are
harvested OR microbe makes protein product and the
protein product is harvested.
1. Isolate target vector
• Plasmid vectors can be obtained from
microbes themselves, or by ordering them
commercially.
– If a the vector is to be obtained from a microbe,
that microbe is grown in mass.
• The the microbes are lysed, releasing their
intracellular contents.
• Then the plasmid is isolated from protein and
membrane debris
– If the plasmid is obtain commercially it means
that it was previously grown in a microbe and
isolated.
• The advantage to buying a plasmid commercially is
that it’s nucleotide sequence is already known. This
is called a map. See Fig. 10.8 for an example.
• The map shows what genes are on the plasmid, the
size, as well as the sites called restriction enzyme
sites.
• The restriction enzyme sites are very important for
gene insertion into the plasmid.
2. Original DNA cleaved by
restriction enzymes
• The next step is to isolate the selected gene that
we want to put into E.coli. So if we use the
insulin example, we want to isolate the gene for
insulin.
• Restriction enzymes are designed to cut DNA at a
specific sequence.
– Restriction enzymes are essentially DNA scissors.
– Restriction enzyme sites are like the little diagram of
scissors on a paper project that indicate that you are to
cut on the dotted line.
– Each restriction enzyme will only cut at the DNA
sequence it recognizes. See Fig. 10.1.
– ON the plasmid map you will see many restriction
enzymes marked. Some examples are:
• EcoRI and RII: restriction enzymes made by E.coli;
• HaeI, II, III: restriction enzymes made by Haemophilus
influenzae;
• SalI: a restriction enzyme made by Salmonella;
• The insulin gene and it’s surrounding genetic
sequence is analyzed to determine which
restriction enzyme sites surround the gene and to
cut it out.
Gel Electrophoresis
• After the insulin gene is cut out, it is still
surrounded by cut DNA and restriction
enzymes. It needs to be isolated so that it
can be inserted into the plasmid vector.
• Gel Electrophoresis is used to separate
selected genes from the rest of the cell
garbage.
• The gel that is used for Gel Electrophoresis
is made up of a molecule called agarose.
• To help you understand what is happening
better let’s talk about “Jello Jigglers”.
– For those of you that don’t know what Jello
Jigglers are, they are shapes made from
concentrated jello.
– In other words, less water is added, meaning
that the sugar and gelatin molecules are more
concentrated giving a stiffer jello. The stiffer
jello allows you to cut shapes out of the jello.
• An agarose gel is similar but instead of
gelatin and sugar, agarose is used to create
the “jello”. The more water that is added,
the less concentrated the agarose is.
• Just like when you make jello, the agarose
and water are heated up first. Then they are
poured into a mold to cool. The molds
come in various sizes but essentially they
are all rectangles that are about 1 ½ inches
thick.
• Once the agarose is poured into the mold a
“comb” is put into the agarose at one end of the
gel. It looks similar to a hair comb but there are
usually only 8 or 10 teeth that are very wide and
flat instead of pointed at the ends.
• When the agarose is set, the comb is removed and
small slits remain where the teeth were. These
slits are called wells and are the place where the
DNA is put into the gel.
• A “ladder” is put into one of the wells. The
“ladder” is commercially made by putting DNA
fragements of various known sizes together.
These DNA fragments help the researcher to know
which piece of DNA in the other wells is the DNA
with the insulin gene. (We know the size of the
gene because we have its sequence.)
• Ok. So we load the ladder in one well and the rest
of the wells we fill with the DNA and garbage.
• Each DNA nucleotide has a phophate group on it.
Remember from the cell membrane that a
phophate group has a slight negative charge. So
the charge on DNA is negative.
• We cover the gel with a buffer that conducts
electricity and then put a negative charge on the
side of the rectangle closest to the DNA in the
wells and a positive charge on the opposite end of
the rectangle.
• This arrangement causes the DNA to move down
the gel away from the negative charge, towards the
positive charge.
• The more agarose in the gel, the more
molecules there are that the DNA has to
move through.
• The smaller pieces of DNA can move
through much more quickly than the large
pieces of DNA.
• The large DNA remains closer to the wells
and the small DNA moves closer to the
opposite end.
• After the DNA has been separated on the
gel it is stained so that we can see where the
DNA is. See Fig. 10.2.
• By using the ladder and comparing the
known sizes of DNA on the ladder to the
known size of the insulin gene, we can find
the insulin gene.
• Once the insulin gene is identified on the
gel it can be excised out of the gel by using
a razorblade. Then the agarose is melted
and the DNA removed and cleaned.
Polymerase Chain Reaction (PCR)
• The last step before the insulin gene can be inserted into
the plasmid vector is PCR.
• PCR is a quick and easy way to make multiple gene copies
(DNA replication). See Fig. 10.6.
• 1. First you take a small plastic tube and put in the insulin
gene. It will be the template.
• 2. Then you add some primers. Remember in DNA
replication that a primer is a hand for the other nucleotides
to hold onto so that they can start replication of the
template. The primers that we use for our gene are
specially made so that they recognize and bind to the
template gene sequence at the 5’ end.
• 3. Then some DNA bases are put into the
tube. (ATCG) DNA can’t be made without
them!
• 4. Then a polymerase is added. Just like in
DNA replication, the polymerase puts the
nucleotides together.
• Now were ready to begin the process of
PCR.
• The first step is to heat the DNA.
– The heat breaks the DNA strands apart.
• Then the sample is cooled and the primers bind to
the template strands. Then the polymerase begins
to attach the nucleotides to make complementary
strands.
• Then the cycle begins again. The sample is heated
up, the strands separate. The sample is cooled and
the primers and polymerase synthesize
complementary strands. This process is repeated
many times to create many gene copies.
• PCR used to be done using water baths heated to
the necessary temperatures. Then a timer would
be set for a couple of minutes and the researcher
would have to move the sample from bath to bath
cycle after cycle. Now days there is a special
machine made just for cycling the temperatures
for PCR
• After the PCR is finished, another round of
gel electrophoresis is performed to get rid of
the enzymes and unused nucleotides.
• Now the gene is read to be inserted into the
plasmid vector.
• It’s the moment you have been waiting for
and you get to wait some more until next
lecture. I hope you’re not too disappointed.
• Homework will be given after the next
lecture.