Recombinant DNA Pape..

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11 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

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Recombinant DNA Paper Plasmid Lab


Use your text book to define the following terms in your lab notebook.

recombinant DNA


genetic engineering


restriction enzyme


plasmid



Background Information


Recombinant DNA technology is one of the new techniques of

biotechnology. Biotechnology uses living
organisms to carry out chemical processes or to produce substances, combining biology with chemistry and
science with industry. Biotechnology includes the field of
genetic engineering,

which is the science of
dir
ectly manipulating genes to produce recombinant DNA. Genetic engineering techniques are used in a
variety of industries, in agriculture, in basic research, and in medicine.


Through genetic engineering, there is great potential for the development of usef
ul products. Researchers
have already developed sources of interferon, human growth hormone, insulin, and resistant fruits and
vegetables. A gene coding for a particular protein is transferred into a host organism. The host organism
reproduces and the p
opulation produces the desired protein in volume. For example, the gene that codes for
the production of human insulin has been inserted into the common bacterium
E. coli
. Then the bacteria can
be grown in huge containers and large amounts of insulin can

be collected.


As an introduction to recombinant DNA technology, the exercise that follows illustrates on paper some of
the steps of recombinant DNA experiments done in laboratories.



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Steps to Recombination


1.

Scientists must first
identify the gene

tha
t codes for the production of the protein they want to


manufacture.


2.

Next scientists must
isolate the desired gene
. Restriction enzymes from bacterial cells are important


in this step. Each
restriction enzyme

recognizes and cleaves (cuts) a very s
pecific sequence of DNA


called a
restriction site
. Some restriction enzymes make a staggered cut of the DNA producing


sticky ends

or single strands of unpaired nucleotide bases capable of binding with complementary


sticky ends. By using restriction
enzymes that will cut the DNA on either side of the gene, the desired


gene can be clipped out of the DNA strand.




3.

Once the desired gene has been isolated scientists must then
insert the gene into a plasmid
.


Plasmids

are small circular pieces of DN
A found naturally in most bacteria. The plasmid must be


removed from a bacterium and cleaved with the same restriction enzyme used to isolate the gene.


This way the sticky ends of the plasmid will match those of the gene.



3

4.

Finally, bioengineers m
ix the plasmids with host bacteria. The
recombinant plasmid is taken up by a


host bacterium
. Inside the bacterium, the plasmids

replicate so that they exist in multiple copies. The


gene becomes active and the
bacteria begin producing the protein
.




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Procedure for Making a Paper Model of Recombinant DNA

1.

Construct a plasmid
.


a.

Cut out the plasmid strips along the dotted lines.


b.

Tape them together, end to end, in any order.


c.

Tape the two ends of the linear piece together to give the plasmid i
ts circular shape.

2.

Map the plasmid
.


a.

Color the restriction site on each enzyme card with a different color.


b.

Cut the enzyme sheet into separate cards.


c.

Mark a starting point on the plasmid by drawing a circle around any base pair.


d.

Locate th
e restriction sites on the plasmid by comparing the sequence of base pairs on the enzyme



cards with the sequence of base pairs on the plasmid.


e.

Mark as many restriction sites as possible by drawing a line along the plasmid restriction site which



ma
tches the line drawn on the enzyme card. Make certain that you use the same color as the



enzyme card. Write the name of the restriction enzyme beside the cleavage site.

3.

Construct the cell DNA and isolate the gene to be inserted into the plasmid
.


a
.

Leaving the numbers 1 through 6 on the bottom of each strip, cut out the strips on the cell DNA



sheet.


b.

Assemble the strips in order by taping the top of strip 2 to the bottom of strip 1. Tape the top of



strip 3 to the bottom of strip 2. Continu
e in this fashion until all 6 strips have been assembled into



a chromosome strand.

4.

Determine which restriction sites are near the protein gene and cleave with the restriction enzyme
.


a.

The protein gene is marked like this:


b.

Choose a restriction e
nzyme that cuts your plasmid in only one place.



Locate and mark that
same

restriction enzyme’s site
above and below

the gene by comparing the



sequence of base pairs on the enzyme card with the sequence of base pairs on the cell DNA.


c.

Cut both the p
lasmid and the cell DNA along the
same

restriction enzyme site. The plasmid will be



cut in one place and the cell DNA will be cut in two places.

5.

Insert the gene into the plasmid
.


a.

Match the
sticky ends

of the gene with those of the plasmid and ta
pe the ends so that the gene



is incorporated into the plasmid.


b.

You now have a recombinant DNA molecule composed of DNA from two different organisms.



At this point in a recombinant DNA laboratory, the recombinant plasmids would be mixed with



the h
ost bacterial cells and could be screened for an antibiotic marker or for the expression of the



inserted gene.