Biology 234
J. G. Doheny
Chapter 10 (Part I)
Gene Isolation and Manipulation
Practice Questions:
Answer the following questions with one or two sentences.
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2.
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5.
From which types of organisms were most restriction enzymes originally isolated?
What is ‘bacterial transformation?’
Which bacteria do people usually transform with plasmid vectors?
Which ‘host’ organism do people usually use for cloning work?
Rank these three types of plasmids in order of size, starting with the smallest: YAC,
Plasmid, HAC. What is the approximate size of the insert that can be put in each?
6. What is a transgene?
7. What is a plasmid (not a plasmid vector)?
8. What is a plasmid vector?
9. What is a bacterial colony?
10. What is a polylinker?
11. What is a multiple cloning site (MCS)?
12. What is ampicillin, and what is it used for?
13. What is kanamycin, and what is it used for?
14. What are Type II restriction enzymes?
15. What are isoschizomers?
16. What is a 6 cutter restriction enzyme?
17. What is a “sticky end?”
18. What is a 5’ overhang?
19. What is a 3’ overhang?
20. What is a blunt-end cutter?
21. What does the enzyme DNA Ligase do?
22. What does the enzyme Mung Bean nuclease do?
23. What is a restriction digest?
24. What is a partial digest (also known as an incomplete digest)?
25. What is a restriction fragment?
26. What is a polyhistidine tag?
27. What is nickel-sepharose?
28. What is an affinity column?
29. What does MCAC stand for?
30. What is an elution profile?
31. What is a fraction collector used for?
32. What is the O.D. of a bacterial culture?
33. What is X-Gal? What is X-Gal an artificial version of?
34. What is a chromagen?
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35. What does β-Gal stand for? What is it?
36. What is a reverse transcriptase enzyme?
37. What is pBR322?
38. What is directional cloning?
39. What is IPTG? (You don’t have to know the full name, just what it’s used for.) What is
IPTG an artificial version of?
40. What is a secretion peptide? (One form of a signal peptide.)
41. Many recombinant proteins are produced in bacterial cells. List three other types of cells
that can also be used to produce recombinant proteins.
42. List the name of one restriction enzyme that generates a 5’ overhang? (Also, list one
restriction enzyme that can generate a 3’ overhang, and one that generates a blunt end.)
43. List one restriction site (for a restriction enzyme) that contains a start codon.
44. List two restriction enzymes that do not recognize the same cutting site, but which will
generate compatible sticky ends after cutting.
45. What is an orbital shaker platform, and what is it used for?
Be able to answer the following questions in one or two paragraphs:
1.
2.
3.
4.
5.
6.
7.
What is the difference between a naturally occurring plasmid, and a plasmid vector?
What is an antibiotic resistance marker?
What is the LacZ gene?
What does the β-Galactosidase enzyme do?
What is blue/white screening, and how is it done?
What is the difference between a genetic selection and a genetic screen?
Briefly explain how antibiotics, such as ampicillin, are used to separate bacteria that have
received a plasmid vector from those that have not.
8. What is the difference between a 6 cutter and a 4 cutter restriction enzyme, and which has
the more common restriction site?
9. When talking about restriction digests, what is an ‘incomplete digest? When would you
use an incomplete digest, as opposed to a complete digest?
10. MATH PROBLEM: Suppose you have a large sequence of DNA (say, several billion
base pairs), which is composed randomly of the four nucleotides A, T, G and C. Now
suppose you have a four cutter restriction enzyme that recognizes the sequence ATTA.
How often would you expect to find this restriction site in this DNA sequence purely by
chance? How about a six-cutter (like EcoRI) that recognizes the sequence GAATTC?
(Hint: use the Product Rule.)
11. What are “sticky ends” generated by restriction enzymes?
12. What is a shuttle vector?
13. What is an expression vector?
14. What is the difference between a cloning vector and an expression vector?
15. Why is it necessary to do “spread plating” when you “plate” transformed bacteria onto a
plate? Why can’t you just pour the test tube of bacteria onto the plate?
16. Explain how an incomplete digest (or partial digest) of genomic DNA can result in a
series of overlapping DNA fragments.
17. Explain how the cuts made to DNA by EcoRI, EcoRV and KpnI differ.
18. Explain two methods by which a restriction enzyme sticky end can be converted to a
blunt end.
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19. Explain one method for measuring the O.D. of a bacterial culture where you do not have
to risk contaminating the culture by opening the culture flask.
20. What is the difference between cloning and subcloning?
Be able to explain the following in one hand-written page or less:
1. Using the AmpR gene and LacZ genes as examples, explain how genetic selections and
genetic screenings work.
2. What is the LacZ gene, and how can it be used to do genetic screens?
3. What is the difference between a cloning vector, a shuttle vector, and an expression
vector?
4. Explain how Blue/White screening works to identify plasmid vectors that have received
an insert into the MCS.
5. What is a serial dilution, and what would you use a serial dilution for in the process of
cloning a gene?
6. Explain how you could convert EcoRI-generated sticky ends into a form that would be
compatible with EcoRV.
7. Explain how a genomic library is made.
8. Explain what a cDNA library is, and how it is made.
9. Explain how ampicillin works to kill bacteria, and how the ampicillin resistance gene
prevents it from doing so.
10. Explain how kanamycin works to kill bacteria, and how the kanamycin resistance gene
prevents it from doing so.
11. Explain why kanamycin can be used to grow up very dense cultures of plasmidcontaining bacteria and ampicillin cannot. (It may be helpful to explain how these two
antibiotics work, as well as how the resistance genes work overcome the effects of the
antibiotics.)
The Recognition Sequences of some Type II Restriction Enzymes.
(Please memorize the names of at least one blunt end 6 cutter, one 6 cutter enzyme leaving a 5’
overhang, and one 6 cutter enzyme leaving a 3’ overhang. Also memorize the name of a
restriction site for a 6 cutter enzyme that contains a start codon. Also, memorize the names of
two DIFFERENT restriction enzymes that generate compatible ends. Also, memorize the name
of one 4 cutter and one 8 cutter enzyme.) Note: the ^ symbol indicates where the sequence is
cut.
NcoI: 5’-C^CATGG-3’
Hind III: 5’-A^AGCTT-3’
EcoRI: 5’-G^AATTC-3’
EcoRV: 5’-GAT^ATC-3’
KpnI: 5’-GGTAC^C-3’
NheI: 5’-G^CTAGC-3’
SpeI: 5’-A^CTAGT-3’
AluI: 5’-AG^CT-3’
AscI: 5’-GG^CGCGCC-3’
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Exercise: Based on these sequences and cutting sites, tell me
A. Which generate a 5’ sticky end, a 3’ sticky end, or a blunt end.
B. Which one of these contains a START codon.
C. Which have compatible sticky ends, despite not having the same recognition site.
Question: It is possible to turn a ‘sticky end’ into a ‘blunt end’ by first cutting the DNA, and
then adding DNA Polymerase and nucleotides to ‘fill in’ the sticky end. Which of the above
enzyme cutting sites could be cut, and then converted into a blunt end using this method? It is
also possible to convert a sticky end into a blunt end by ‘chewing away’ either a 5’ or a 3’
overhang with Mung Bean Nuclease. Which of the above enzyme cutting sites could be
converted to a cut and then converted to a blunt end this way?
Restriction Enzyme Problem:
Look at the plasmid maps in the figure below. Let’s build an expression plasmid where the
human insulin gene (created from a cDNA) is put into a pUC19 cloning vector (Vector Construct
A). The bacterial secretion peptide for BOTOX, the neurotoxin secreted by Clostridium
botulinum is cloned into another pUC19 cloning vector (Vector Construct B). Use restriction
enzymes (and any tricks you’ve learned about using them) to clone the BOTOX secretion
peptide (SP) onto the amino terminus of the insulin gene, and then put this fusion gene into the
pET27b expression vector. The pET27b expression vector will then put a His6 affinity tag onto
the carboxyl terminus of the recombinant protein. If you are successful in doing this, you will be
able to produce human insulin in E. coli. The insulin will be secreted into the bacterial growth
medium (thanks to the secretion peptide), and you’ll easily be able to purify the insulin using the
His6 affinity tag, and MCAC. How would you do the cloning?
Note: the insulin gene is 2kb in size, the BOTOX SP is 1kb in size, and the distance between the
NheI and HindIII sites in the MCS (Polylinker) pET27b is 0.5kb. Remember that pUC19 is 2kb
in size, and pET27b is 1kb in size. Start by calculating the total sizes of these vectors when
the gene inserts are inside. Also, for purposes of this exercise, don’t worry about splicing the
genes together in the right reading frame. Assume that when you put these DNA fragments
together, they will already be in the correct reading frame.
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Recommended questions from the textbook.
3, 5, 6, 14, 15, 18, 20, 21, 32
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