Bio 201 Spring 2014
Ch. 9 Part 1 Homework
Name: _____________________________
Use the information and diagrams below to answer questions 1-10 (5 points each).
You are employed at a pharmaceutical company trying to generate massive amounts of Protein A from Ascary bacterium, which will be used in a vaccine to treat Anasty disease which has been on the rise (yes, if you are wondering I made this up!). First you isolated DNA from Ascary bacteria.
1.
You then decide to use the restriction enzyme BamHI on the isolated DNA. Below is the DNA sequence of a section of Ascary DNA. Indicate with arrows where BamHI will cut this DNA on both strands.
5’ ATATGCAG^GATCCGCATA 3’
3’ TATACGTCCTAG^GCGTAT 5’
2.
(Circle one) What type of ends will be generated in question 1? (sticky, blunt, or both?)
Because you treated the entire DNA from Ascary bacterium with the restriction enzyme in question 1, you need to gel electrophoresis the DNA samples. Below are your results:
3.
The gene encoding Protein A from Ascary bacterium is 2kb long. Which sample in this diagram contains this gene?__________2_______________
4.
Based on question 3, circle the band gene encoding
Protein A from Ascary bacterium on the gel.
5.
Label on the gel the positive and negative ends. – at top, + at bottom
6.
What must be added to the gel in order to visualize the DNA?__________Ethidium bromide/intercalating agent_____________
7.
Now that you have a purified gene encoding Protein
A from Ascary bacterium, you want to clone it to have it replicate in a new host in large amounts. You decide to use a plasmid as the vector for cloning. Based on question
1, what enzyme must you use to cut the vector/plasmid?______BamHI_____________
8.
(True or false): The DNA containing the gene which encodes Protein A (DNA only not DNA and vector) from Ascary will integrate into the chromosome by homologous recombination during cloning.
9.
(True or false): The DNA containing the gene which encodes Protein A (DNA only not DNA and vector) from Ascary does not contain an origin of replication.

10.
Based on question 7, the recombinant DNA in this entire experiment is: a.
The vector alone b.
The DNA encoding Protein A from Ascary bacterium c.
The vector and DNA encoding Protein A from Ascary bacterium joined together d.
None of the above
BONUS! (10 points)
Because you were so successful at using biotechnology techniques to produce large quantities of
Protein A from Ascary bacteria, you were moved to another department at the company, which is trying to isolate large quantities of human growth hormone (HGH) to treat dwarfism. Describe how you would go about cloning the human growth hormone gene in E. coli . You have the following tools: a probe, any vector you would like, human cells, and E. coli . Please note we have not gone into specific details about the probe. You can be general when describing that in your methods. Also complete sentences are not needed. You can describe it as a method/procedure approach in a lab exercise manner.
Construct a plasmid library containing human cDNA

Screen the library for the hgh gene with the probe/selectable market.
Construction of the library:
1.
Make cDNA from human cell mRNA using reverse transcriptase
2.
Isolate plasmid DNA for vector from bacteria.
3.
Digest cDNA and plasmid DNA with the same restriction enzymes.
4.
Ligate the sticky ends of the restriction enzyme digested cDNA from human cells and plasmid DNA together using DNA ligase.
Transform the recombinant DNA molecules into E. coli for amplification of the DNA. Plate the transformed
DNA onto agar plates containing the antibiotic to which the plasmid confers resistance. Each colony came from one cell that contains a recombinant plasmid that contains one fragment of human cDNA.