BIOL 1010
LECTURE ASSIGNMENT
RECOMBINANT DNA EXERCISE: PLASMIDS
Introduction:
Chapter 12 of your textbook does a rather good job of explaining and diagramming the
utilization of plasmids in recombinant DNA procedures.
Recombinant DNA technology is a means by which scientists can insert genes from one
species, into the DNA of another. The classic example of recombinant DNA technology
is where the human insulin gene was isolated from human DNA, and was then inserted
into a bacterium, using a plasmid as a vector (see next paragraph) – the bacterium could
then produce the human form of insulin.
A plasmid is a circlet of DNA found in a bacterium. Plasmids are unique to bacteria and
are a means by which bacteria can actually exchange genetic material. Scientists have
determined that plasmids are an excellent means of moving desirable genes into bacterial
cells (see Fig. 12.1 on page 233); these bacterial cells can rapidly divide, thus producing
numerous “clones” or genetically identical copies. If the gene codes for a desired protein
(like insulin), all you have to do is grow the bacteria in large numbers – they crank out
the protein (insulin)!
This exercise is a fun one – all the materials and instructions are obtained from an
internet URL; the student gets to play with scissors and tape and ultimately gets an
excellent idea of the process of using plasmids to genetically engineer bacteria.
Specifically, you will need to choose a restriction enzyme that will cut a human insulin
gene out of a human DNA molecule. The same restriction enzyme will be used to open a
circular plasmid, allowing the insulin gene to be spliced in. Ideally, if this plasmid were
to be successfully introduced into a bacterium, the bacterium should be able to produce
human insulin; many clones of this bacterium could result in the production of a large
amount of human insulin – perfect for diabetic patients who do not make enough insulin
of their own.
READ THE INSTRUCTIONS CAREFULLY!
Instructions:
1. Using your browser’s address box, go to
http://www.accessexcellence.com/AB/WYW/wkbooks/SFTS/activity6.html. [If
you are having difficulty getting to this URL, let me know ASAP]
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2. Read Objective on web site.
3. Read Background Information on web site – make sure that you understand the
concept of “sticky ends”.
4. Read Materials on web site. [NOTE: YOU WILL BE DOING THIS EXERCISE
INDIVIDUALLY] The Handouts mentioned here are accessed by clicking on
their names.
5. You will need to print each of the three Handouts listed on the web site.
The following steps are modified from the Instructions found on the web site –
THE STUDENT SHOULD FOLLOW THE INSTRUCTIONS THAT FOLLOW
HERE!
6. Cut the Plasmid Base Sequence Strips (Handout 3) along the dotted lines, shuffle
the strips and tape them together to form a long strip. Be sure that the letters are
oriented in the same direction all along the strip. Tape the two ends of the strip
together to form one circular plasmid – be sure that the base sequences of the
plasmid are facing out. [Note: This is the plasmid BEFORE you perform the
recombinant procedure; this plasmid is destined to carry the insulin gene that you
want to insert into your bacterium].
7. Cut the DNA Base Sequence Strips (Handout 4) along the dotted lines and tape
them together in the order indicated to make one long strip. [Note: this strip of
human DNA includes the gene for insulin (shaded regions on strips 3, 4, and 5) –
this is the gene to be cut out for insertion into the plasmid].
8. Cut the Restriction Enzyme Sequence Cards (Handout 5) along the dotted lines.
Each of these cards shows the particular “cut” that a certain restriction enzyme
makes in a DNA sequence. [Note: the different restriction enzymes make very
specific cuts at very specific DNA sequence sites.
9. Take each Restriction Enzyme Sequence Card and compare it to your plasmid’s
sequence. Find the place on the plasmid where the enzyme will make a cut, mark
that place with a pencil, and write in the appropriate number of the enzyme on the
plasmid. You should then make sure you know which of the restriction enzymes
cuts the plasmid once and only once (why would this be important?). [Note: for
our purposes, we will ignore the shaded region of the plasmid sequence].
10. Take the same Restriction Enzyme Sequence Cards and compare each to the DNA
molecule. Which restriction enzymes will cut the DNA molecule both above and
below the insulin gene? Mark the location as you did for the plasmid and indicate
the identity of the enzyme.
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11. Now it is time to choose the best restriction enzyme. The best choice will cut the
plasmid in only one place. The best choice will also cut the DNA molecule both
above and below the insulin gene, as closely as possible without cutting the
insulin sequence itself.
12. Once you have made your choice, make the appropriate cuts to your plasmid and
your DNA molecule (use the same staggered cuts indicated on the Restriction
Enzyme Sequence Card).
13. Using clear tape, splice the insulin gene into the plasmid.
14. You are finished! Almost . . . All you have to do now is to . . .
15. Fold your recombinant plasmid so that it will fit into a business-sized envelope
[NOT a manila envelope]. Put the plasmid AND your chosen Restriction Enzyme
Sequence Card in the envelope ALONG WITH answers to the following
questions answered [typed please!]:
a. Why would you want your restriction enzyme to cut the plasmid in only
one location?
b. Why would you want your restriction enzyme to cut as closely to the
insulin gene as possible without cutting into it?
c. After doing this exercise – do you think genetic engineering of this sort is
an easy process or not. Why?
MAKE SURE YOU ANSWER ALL 3 QUESTIONS ADEQUATELY!
BELIEVE ME . . .
IN YOUR OWN WORDS – NO CUT-AND-PASTE STUFF . . .
YOU DO NOT WANT TO SCREW THIS UP!
16. Seal the envelope and write your name on it – be sure to hand it in to Miss
Blumke on or before 10/8/2009! Late submissions will not be accepted [ie. DO
NOT HAND IT TO ME AT THE FINAL EXAM!]
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GRADING SCALE: OUT OF 100 POINTS
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Plasmid not assembled correctly
Plasmid missing
Insulin gene not inserted into the plasmid correctly
DNA sequence not assembled correctly
DNA sequence missing
Wrong restriction enzyme
Restriction enzyme missing
Wrong answers to three questions (see #15 above)
Answers not typed
No answers at all
Inappropriate envelope
No name on envelope
minus 5 points
minus 10 points
minus 5 points
minus 5 points
minus 10 points
minus 10 points
minus 10 points
minus 5 points each
minus 5 points
minus 15 points
minus 5 points
minus 5 points
PLEASE NOTE: THE GRADING SCALE IS A GOOD INDICATION OF HOW
IMPORTANT IT IS THAT YOU FOLLOW THE INSTRUCTIONS CAREFULLY!
ADDITIONAL NOTE: IF I DETECT ANSWERS THAT ARE COPIED FROM
ANYONE ELSE, I WILL AWARD A 0 TO EACH INDIVIDUAL INVOLVED – THIS
SHOULD BE INCENTIVE TO DO YOUR OWN WORK!
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