HUMAN INSULIN GENE

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BIOL 1010 OPENSTAX PROJECT INSTRUCTIONS

Introduction. This BIOL 1010 OpenStax and LibGuides Project specifically concerns the topic of Genetic Engineering. The project should be completed in accordance with the requirements contained in this document. The Grading Rubric for the project is included at the end of this document to maximize your grade for this assignment.

The student should be careful to make sure that all directions are followed in completing the assignment.

MODELING RECOMBINANT DNA: HUMAN INSULIN GENE

Introduction. The manufacture of human insulin is a genetic engineering success story. Prior to the 1980’s diabetes was treated with insulin extracted from the pancreas glands of cows and pigs. While the animal-derived insulin was effective in treating diabetes, it was not structurally identical to human insulin; side effects and allergic reactions were not uncommon problems. In 1981, two U.S. companies, Genentech and Eli Lily, succeeded in inserting the human insulin gene into E. coli (Escherischia coli, a bacterium). Later, a Danish company, Novo Nordisk was able to genetically modify yeasts (single-celled fungi) for the purposes of producing human insulin. Diabetes treatment in more developed countries today is dominated by human insulin produced by genetically engineered bacteria or yeasts.

This project will model the process of genetic engineering that led to the production of human insulin by E. coli bacteria. You will be using printed paper strips to represent the DNA sequence that codes for human insulin and the bacterial plasmid into which the human insulin gene will be spliced. You will be provided with a selection of restriction enzymes that could be used to cut and splice these components. Your task will be to find the one restriction enzyme that makes the appropriate cuts so that you can splice the human insulin gene into the bacterial plasmid. You will need some simple materials to complete this project (below). Content background for this project will be found in your OpenStax textbook (chapters 9 and 10) and the BIOL 1010 LibGuides (Khan Academy pages on Molecular Biology and Biotechnology) pages at http://getlibraryhelp.highlands.edu/.

Materials needed:

White paper for printing

Colored paper (preferably a lighter color) for printing

Clear tape

Business-sized envelope

Highlighter marker

Scissors

Ruler

Preparation. Before you start your project (instructions under Task below), you will want to become familiar with the process of recombinant DNA using restriction enzymes and plasmids. Be sure that you use the following as resources:

OpenStax Concepts of Biology, chapter 9.

OpenStax Concepts of Biology, chapter 10.

LibGuide (Molecular Biology, Khan Academy) at http://getlibraryhelp.highlands.edu/

LibGuide (Biotechnology, Khan Academy) at http://getlibraryhelp.highlands.edu/

Task.

Your ultimate goal: generate a bacterial plasmid that contains the entire human insulin gene. There are other conditions that must be met for success. Read the instructions carefully! Your ability to follow instructions will be critical to your success! All necessary files for printing the DNA sequence, plasmid sequence, and restriction enzymes will be found on D2L in the OpenStax Project folder.

Step 1. Assemble the DNA sequence. You will generate a paper model of a human DNA sequence that contains the human insulin gene.

A. Print the DNA SEQUENCE pages (source: D2L) on colored paper (preferably a light colored paper) – the color is your choice. You will notice that the DNA sequence consists of A’s, C’s, G’s, and T’s in pairs. In other words, there are two parallel strands of nucleotides, one is the template strand, and the other is the coding strand. The sequence is oriented vertically and each strand is read from top to bottom.

B. Using a ruler, draw parallel lines vertically so that each sequence can be cut into ¾” wide strips; you want your DNA SEQUENCE to look nice-and-neat when you are finished.

C. Cut out the ¾” strips that are found on the DNA SEQUENCE pages.

D. Tape the strips (10 of them) together in order (as shown below).

1 2 3 4 5 6 7 8 9 10

¾”

[Be sure to tape the strips so that the sequence is continuous; the strip numbers and the 3’/5’ designations should not show when two adjoining strips are taped

together…the entire strip should be an uninterrupted series of A’s, C’s, G’s, and T’s in pairs.

E. Note that the human insulin gene is represented by the bold print sequence on the strip. Your completed DNA SEQUENCE should contain the bold print insulin gene flanked on either side by “unbold” sequences.

Step 2. Assemble the plasmid sequence. You will generate a paper model of a bacterial plasmid.

A. Print the PLASMID page (source: D2L) on white paper. You will note that the plasmid sequence looks just like the DNA sequence in Step 1.

B. Using a ruler, draw parallel lines vertically so that each plasmid sequence can be cut into ¾” strips. You will notice four bracketed abbreviations on your plasmid sequence. While these are not critical to your final presentation, you will want to make a deviation in your cutting so these bracketed abbreviations stay on your plasmid.

C. Cut out the strips that are found on the PLASMID page and tape them together (the order of the strips is not important) to form a circle.

Step 3. Obtain your restriction enzymes. You will print and cut out 8 different restriction enzymes. One of these enzymes will be chosen to cut the plasmid and the insulin gene so that the insulin gene can be spliced into the plasmid.

A. Print the RESTRICTION ENZYME page (source: D2L)… the color of paper does not matter.

B. Cut out the individual enzymes…you should have a total of 8 individual enzymes. You will note that each RESTRICTION ENZYME makes a cut (dotted line) associated with a specific sequence of nucleotides.

Step 4. Marking where the restriction enzymes cut the DNA and the plasmid. It is time to determine which of the 8 restriction enzymes will be able to work for you in genetically engineering this bacterium. You will begin by marking where each restriction enzyme will cut the DNA sequence (specific instructions are found in steps A-D. Then, mark the plasmid in the same way for each of the 8 restriction enzymes. [Your restriction enzyme cards will be used as a guide for marking where the cuts will occur on both the DNA SEQUENCE strip and the PLASMID; the dotted line on each restriction enzyme card indicates where the cut is to be made].

HINT: STEP C IS CRITICAL TO YOUR SUCCESS AT THIS POINT!

A. Take your DNA SEQUENCE strip and mark the locations of cuts for each of your 8 RESTRICTION ENZYMES (use a pencil – be sure to identify the cut with the name of the enzyme on the DNA SEQUENCE strip).

B. Take your PLASMID (circle) and mark the locations of cuts for each of your 8 RESTRICTION ENZYMES (use a pencil – be sure to identify the cut with the name of the enzyme).

C. Look carefully at the cut locations on your DNA SEQUENCE and PLASMID. You need to identify the one RESTRICTION ENZYME that both

a. Cuts the PLASMID at only one location, and

b. Cuts the DNA SEQUENCE strip on either side of the insulin gene without cutting into the insulin gene.

D. Be sure to keep the appropriate RESTRICTION ENZYME; do not lose it. The appropriate RESTRICTION ENZYME will be submitted with the completed project.

IMPORTANT: Please be aware of the fact that some of the restriction enzymes will not work. You need to be concerned with marking the locations of the cuts for the restriction enzymes that do work!

Step 5. Cut and splice time. Using your specific RESTRICTION ENZYME identified in Step 4 as a template, you will make a single cut in the PLASMID and two cuts in the DNA SEQUENCE. Make your cuts carefully! The Human Insulin Gene (cut from the DNA SEQUENCE) will then be spliced into the cut PLASMID.

A. Make the appropriate cuts identified in Step 4. C above. You will note that the cuts make “sticky ends” that will be complementary to the other cut ends. Here is an example of how two sticky ends can be joined together in a complementary DNA sequence (below – note that the two sticky ends join in such a way as the base pair combinations CG/AT are maintained).

AGTC + CGGTACCGTAC AGTCCGGTACCGTAC

TCAGGCCAT GGCATG TCAGGCCATGGCATG

sticky end sticky end sticky ends joined together

B. Open the PLASMID and splice the cut ends of the DNA SEQUENCE strip into the PLASMID. Use tape to fix the splices in place. You have created a RECOMBINANT PLASMID. Your result should be a circle of DNA that includes the original PLASMID (white strip) and the DNA SEQUENCE (colored strip) featuring the complete Human Insulin Gene (in bold print).

Step 6. Get ready to hand in your genetically engineered plasmid. A portion of your grade depends on you following these directions carefully.

A. Carefully fold your RECOMBINANT PLASMID so that it will fit into a #10 standard business-sized envelope. It must be folded neatly!

B. Do not seal the envelope.

C. Tape the appropriate RESTRICTION ENZYME TO THE BACK OF THE ENVELOPE and write your name on the front of envelope.

Step 7. Answer Questions 1-7. Questions 1-6 should be submitted as one hardcopy document with “BIOL 1010 OpenStax and LibGuides Project: Questions 1-6” as the title. Question 7 will be submitted as a separate document (see instructions for Question 7 below).

1. What are plasmids? Where are they found? Why are they important to the practice of genetic engineering?

2. Do plasmids have an importance beyond the practice of genetic engineering? Explain.

3. What are restriction enzymes?

4. You might wonder why we might have and origin of replication indicated on the plasmid. What is the origin of replication and why is it important to the genetic engineering process?

5. You might wonder why there are antibiotic resistance genes in the plasmid [genes that codes for resistance to specific antibiotics). Hint: “The antibiotic resistance genes will be used for screening purposes.” What could this mean? Explain.

6. Why would you want your restriction enzyme to cut as close as possible to the insulin gene without cutting into it?

7. [IMPORTANT: YOUR RESPONSE TO THIS QUESTION #7 WILL BE SUBMITTED AS A SEPARATE DOCUMENT] The Human Insulin Gene is a sequence of DNA that ultimately codes for amino acids of specific identities in a specific order. Since DNA is a double-stranded molecule, there are two complementary sequences present: the template strand and the coding strand. From the Human Insulin Gene sequence in your RECOMBINANT PLASMID, you should be able to generate a list of the appropriate amino acids in the correct order. All you will need is a codon chart; a chart that cross-indexes codons with amino acids (OpenStax Concepts of Biology, Figure 9.20, p.220). You will submit this response (#7) on a separate piece of paper with “#7 HUMAN INSULIN: AMINO ACID SEQUENCE” centered at the top of the page. The amino acids should be listed in a vertical column (or columns, if needed). Be sure to list the amino acids by their standard three-letter abbreviation. For example, your first amino acid will be Met (methionine).

Step 8. Handing it all in! You will need a large (10 x 13) manila envelope. On the front of this envelope, you will print:

· Your name, and

· BIOL 1010 OpenStax and LibGuides Project.

You will place in this envelope:

· Your #10 standard business-sized envelope containing the engineered plasmid,

· Your document: BIOL 1010 OpenStax and LibGuides Project: Questions 1-6, and

· Your document: “#7 HUMAN INSULIN: AMINO ACID SEQUENCE”.

Do not seal the large envelope. Just be sure that the three required items (above) are placed securely and completely inside.

Submit the labeled 10 x 13 envelope and its contents to your instructor on the date indicated in the course syllabus.

THE OPENSTAX PROJECT GRADING RUBRIC

10 x 13 manila envelope 1 _____

…appropriately labeled (per instructions) 1 _____

Business-sized envelope with name 1 _____

Restriction enzyme taped to back of envelope 1 _____

Envelope not sealed 1 _____

Recombinant plasmid in envelope 1 _____

Recombinant plasmid folded 1 _____

Recombinant plasmid a complete circle 1 _____

Restriction enzyme chosen is correct 3 _____

Recombinant plasmid contains insulin gene 3 _____

Insulin gene is complete 3 _____

Splices are consistent with the enzyme’s sequence 3 _____

Questions 1-6 separate document 1 _____

Title for Questions 1-6 (per instructions) correct. 1 _____

Question 1 10 _____

Question 2 10 _____

Question 3 10 _____

Question 4 10 _____

Question 5 10 _____

Question 6 10 _____

Question 7 (Human Insulin Gene: Amino Acid Sequence)…

Separate document 1 _____

Title for Question 7 (per instructions) correct 1 _____

Amino acid list present 1 _____

Amino acid list in a vertical column 1 _____

Amino acid abbreviations present 1 _____

Amino acid abbreviations appropriate 1 _____

First amino acid is correct 3 _____

Amino acid sequence is correct 3 _____

Last amino acid is correct 3 _____

Appropriate number of amino acids 3 _____