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Unit 4.1 Quest: Manufacturing Human Proteins
4.1.1: All About Insulin
1. Determine if each statement applies to Type 1 Diabetes, Type 2 Diabetes, or both types of diabetes. (4 pts)
a. Body does not respond to the secreted insulin. ____Type 2 Diabetes ___
b. Condition of chronic hyperglycemia. ____ BOTH___
c. Treated with insulin. __ Type 1 Diabetes or BOTH _____
d. Body does not make enough insulin. ____ Type 1 Diabetes ____
2. Billy is feeling tired and sluggish. A blood glucose level test, indicates low glucose levels. The reason why is blood
glucose levels are low is because Billy’s body is not producing enough ___________. (2 pts)
A. Glucose
B. Glucagon
C. Insulin
D. T3/T4 (thyroxine)
3. Scientists have tried many ways to create insulin, since 1980s what is the leading source of insulin? (2 pts)
A. Purified horse insulin
C. Chemically synthesized
B. Genetically engineered using yeasts
D. Genetically engineered using bacteria
4. What is one medical intervention for Type 1 Diabetes? What is one intervention for Type 2 Diabetes? (2 pts)
Diagnosis/Treatment/Prevention options
T1D – exercising, take insulin, treatment options, eating less carbs/fats/etc
T2D - exercising, take insulin, take medication, treatment options, eating less carbs/fats/etc
5. A 48-year-old man with Type 2 Diabetes wants to begin an exercise program. He has had diabetes for 8 years, takes
no medication, monitors blood glucose twice a day, has no complications from diabetes, is 130% of ideal body
weight, and follows a 1600 calorie meal plan. What adjustments to food intake, if any, should be suggested? (2 pts)
A. He should carry a fast-acting carbohydrate with him.
B. He should increase his diet by 300 calories to prevent hunger during exercise.
C. He should increase his carbohydrate intake before exercising.
D. There should be no change in diet.
6. Explain three items indicated in the diagram. (6 pts)
Blood sugar is lowest right before 7PM.
Insulin levels are highest around 4PM
Urine tests are negative for glucose at 7pm and 7am
Blood sugar is highest around 8:30pm
Insulin is injected at 7AM
Urine has 5% sugar at 1pm and 10pm
7. What are two ways insulin be administered? (2 pts)
Injections, Insulin pens, Insulin jet injectors, External insulin pumps, Internal insulin pumps, The insulin patch
Or
Long-acting, short acting, intermediate insulin
4.1.2: Protein Factories
8. What is the relationship between genes, traits and proteins? (2 pts)
A. Genes→ proteins→ traits
C. Proteins→ genes → traits
B. Traits→ genes → proteins
D. Genes→ traits→ proteins
9. Which of the following is NOT a reason why plasmids are a helpful tool in genetic engineering? (2 pts)
A. Plasmids are small and can be easily engineered to insert DNA fragments from other organisms
B. Plasmids are naturally occurring in bacteria and do not have to be “made”
C. Plasmids are not capable of replicating on their own
D. Plasmids can be grown in lab environments and be used to produce other substances needed for medical
research or products
10. What does the figure show? (2 pts)
A. Gel electrophoresis – sticky ends
B. Restriction enzymes – blunt ends
C. Restriction enzymes – sticky ends
D. DNA Ligase – sticky ends
11. Put the following steps of bacterial transformation into the correct order. (2 pts)
A. prepare target DNA
D. Insert DNA into the plasmid
A. C, D, A, E, B, F
B.
B. Insert plasmid back into the cell
E. Plasmid multiply inside of the cell
A, C, B, E, F, D
C. C, A, D, B, E, F
C. Isolate the gene
F. Cells produce proteins
D.
A, C, D, B, E, F
12. The plasmid pGLO has several interesting genes engineered into it. Which do you think is the most important gene
for the bacteria on the plasmid? Defend your answer. (4 pts)
Depends on explanation.
Ori – origin of replication, to make additional copies of plasmid
Bla – enzyme ensuring ampicillin resistance
AraC – regulates GFP transcription
GFP - glows
13. What is the difference between the pGLO plasmid and GFP? (2 pts)
pGLO is a plasmid, GFP is a protein or GFP gene is a gene and not an entire bacterial plasmid.
14. Suppose a bacterial culture were mixed with recombinant plasmids containing a gene for resistance to penicillin. The
bacterial culture was then treated with penicillin. Which of the following statements is true? (2 pts)
A. The penicillin will kill the bacteria that were transformed.
B. The gene for antibiotic resistance is not expressed in the bacteria that survive.
C. Those bacteria that are successfully transformed will glow under UV light.
D. Those bacteria that contain the plasmid will survive.
15. Which answer best represents why the E.coli glowed on the LB/amp/ara plates? (2 pts)
A. The B lactamase on the plasmid allowed the growth in the presence of ampicillin
B. The GFP on the plasmid was what glowed green
C. The GFP was expressed due to the presence of arabinose in the plate
D. The araC on the plasmid made the E.coli glow green
16. Which transformation procedure step neutralizes the negative charge on both DNA and phospholipids? (2 pts)
A. Calcium Chloride Treatment C. Incubation on Ice
B. Heat Shock
D. Incubation with LB Broth
17. Which transformation procedure step slows the movements of the plasma membrane lipids? (2 pts)
A. Calcium Chloride Treatment
B. Heat Shock
C. Incubation on Ice
D. Incubation with LB Broth
18. Which component of the pGLO plasmid controls gene expression? (2 pts)
A. Ara C region
C. Origin of replication
B. Beta-lactamase gene D. GFP gene
19. What does it mean to make bacterial cells competent? What is necessary for this process? (3 pts)
Able to take up DNA – calcium chloride (CaCl2) and or heat
20. Explain why you used a LB/ampicillin plate and a LB/ampicillin/arabinose plate to test the +pGLO bacteria. (4 pts)
The LB/ ampicillin plate was used to show that the +pGLO bacteria had taken up the plasmid and to make sure
only the +pGLO bacteria were growing. Bacteria without ampicillin resistance would not be able to grow.
The LB/Ampicillin/arabinose tested that the +pGLO bacteria had taken up the plasmid and that they could
produce the GFP protein which allowed them to fluoresce. The arabinose is present, which makes the araC gene
activate the GFP gene to make the GFP protein, which fluoresces.
21. In a microbiology lab, a student completes a bacterial transformation
procedure to introduce a plasmid with a gene for kanamycin resistance to E.
coli bacterial cells. (Kanamycin is an antibiotic). Which plate will grow bacterial
cells that were successfully transformed? (2 pts)
A. Plate I
B. Plate II
C. Plate III
D. Plate IV
22. Which transformation step allows the beta-lactamase production? (2 pts)
A. Calcium Chloride Treatment
B. Heat Shock
C. Incubation on Ice
D. Incubation with Nutrient Broth
23. In the pGlo experiment, why was a plasmid with ampicillin resistance used? (2 pts)
A. We wanted to make sure that even if the bacteria died from the ampicillin, the plasmid would continue to live.
B. It ensured nothing else would grow on the plate except for the E. coli that had the plasmid inserted.
C. The ampicillin activated the metabolic pathway that turned on the GFP gene.
D. The purpose of the experiment was to grow ampicillin resistant E. coli.
24. You were absent from class on the day we did the pGLO
transformation lab. Unfortunately, your lab partner did not label
the plates very well (ok…no labels at all!!!) and he is gone today.
You are trying to decipher the results (assume the transformation
was successful). Based on your predictions and the data you
collect from the plates, determine which plate is which? (4 pts)
___B__ Plate 1
A. LB/AMP + pGLO
___D__ Plate 2
B. LB/AMP/Arabinose + pGLO
___C__ Plate 3
C. LB/AMP -pGLO
___A__ Plate 4
D. LB –pGLO
1
2
3
4
25. pGLO has been transformed into bacteria. Why colonies glowing green? expressing GFP gene, GFP is made (2 pts)
26. Given the following data, calculate the transformation efficiency: (8 pts)
Calculate the following to help you answer Part A:
DNA (µg) =
(Concentration of DNA (µg/µl) x (Volume of DNA in µl)
Total Amount of DNA (µg)
Used in Experiment = _0.09 ug/ul__
Fraction of DNA =
Volume Spread on LB/amp Plate ÷
Total Volume in Test Tube
Fraction of DNA = ___100 / 310 = .32_
pGLO DNA Spread (µg) =
Count Total Number of Cells __19__
Total Amount of DNA Used (µg) x Fraction of DNA
Givens:
pGLO DNA Spread (µg) = _0.09 x 10 x .32 = .288 = .29 ug__
Volume of DNA = 10 µl
pGLO (DNA) Concentration= 0.09 µg/µl
Total Volume in Test Tube = 310 µl
Volume Spread on LB/amp Plate = 100 µl
a) Transformation Efficiency = Total # of Bacterial Colonies on Agar Plate ÷ Amount of DNA Spread on Agar Plate
Show Work:
19 ÷ 0.29 µg = 65.5 transformants/µg = 6.5 x 101 transformants/µg
b) Biotechnologists are in general agreement that the transformation protocol that you have just completed
generally has a transformation efficiency of between 2.0 x 102 and 3.0 x 103 transformants per microgram of DNA.
What does this mean in terms of your laboratory success?
Lab was not successful. The transformed yield was less than 50% of expected. The calculated efficiency (65.5
transformants/µg) is below the predicted limits (200 – 3000 transformants/µg) of efficiency for this protocol.
4.1.3: Protein Purification
27. In 4.1.3, we built a sample protein molecule. What are the four rules that designate how a protein folds? (6 pts)
1. Hydrophobic and nonpolar amino acids = inside
2. Hydrophilic and polar amino acids = outside
3. Charged amino acids (basic = positive, acidic = negative) are outside and form salt bridge bonds with each
other to neutralize charges
4. Cysteine sidechains form a covalent disulfide bonds to stabilize the protein.
28. Which of the following contributes to a proteins’ function? (2 pts)
A. Properties of amino acids that make up the protein
B. Order of amino acids
C. Tertiary structure of the protein
D. A and B only
E. All of the above
29. Hydrophobic interaction chromatography (HIC) separates test preparations based upon __________. (2 pts)
A. Protein shape
C. Location of acidic side chains
B. Location of hydrophobic side chains
D. Salt concentrations
30. Which statement best explains the role of lysozyme and freezing in the GFP chromatography experiment? (2 pts)
A. It helped E.coli grow
C. It ruptured the bacterial cells to release the protein
B. It stabilized E.coli during freezing
D. It stabilized the cells for purification
31. During the final stages of hydrophobic interaction chromatography different buffer solutions were used with
different salt concentrations. Why are the different buffers used to purify the GFP? (2 pts)
A. The buffer solution causes the hydrophobic side chains to move and become attracted to the ions in the buffer.
B. The buffer solution causes the hydrophobic side chains to move and become attracted the hydrophilic matrix in
the HI column.
C. The buffer solution causes the hydrophobic side chains to move and become attracted to the hydrophobic
matrix in the HI column.
D. The buffer solution causes the hydrophobic side chains to move and become attracted to the proteins in the
buffer.
32. Using the protein diagram below, which color (white or dark) do you think are the
hydrophobic amino acids? Why? (2 pts)
Black - hydrophobic amino acids and proteins ‘hide’ on the inside
33. How does amino acid structure relate to the overall shape of a protein? (2 pts)
A. The shape of the protein determines its function
B. The sequence of amino acids determines the shape of the protein
C. The hydrophobicity of each amino acid determines the shape of the protein
D. Proteins are made of nucleic acids, so the amino acid shape does not affect the protein
34. Why does a salt buffer cause hydrophobic amino acids, like the ones in GFP, to push to the outside of a protein and
bind to a chromatography column? (2 pts)
A. The hydrophilic amino acids in GFP push to the inside of the column to be washed out
B. The hydrophobic amino acids in GFP push to the inside of the column to be washed out
C. The hydrophilic amino acids in GFP push to the outside of the protein and bind to the column, while the
hydrophobic proteins can be washed out of the column
D. The hydrophobic amino acids in GFP push to the outside of the protein and bind to the column, while the less
hydrophobic proteins can be washed out of the column
35. HIC and GFP chromatography: (6 pts)
a. Put the tubes in the correct order. (“Step:”)
b. What salt concentration is the buffer? (low, medium or high)
c. What is the name of the buffer?
d. What protein will be collected?
Step: 2
HIC Buffer Name: Wash
Salt concentration: Medium
Step: 3
HIC Buffer Name: Elute
Salt concentration: Low/No
Step: 1
HIC Buffer Name: Bind
Salt concentration: High
Protein collected:
Protein collected:
Protein collected:
Medium/mild
Hydrophobicity
Strong
Hydrophobicity
Hydrophilic
36. Which buffer was used to change the configuration of the GFP protein so that the hydrophobic amino acids faced
the outside of the protein? (2 pts)
A. Elution buffer
C. Wash buffer (medium salt concentration)
B. Binding buffer
D. Equilibrium buffer
4.1.4: Protein Gel Electrophoresis
37. Protein electrophoresis differs from DNA electrophoresis because protein electrophoresis _______. (2 pts)
A. Separates by length
B. Separate by mass
C. Separate by charge
D. All of the above
38. A single protein was denatured and electrophoresed. After visualization, 4 bands were noticed. Two were small and
two were large. What can you conclude from these results? (2 pts)
Answer: A – denaturing with SDS makes sure the proteins no longer
A. Four polypeptides made up the protein
have secondary, tertiary or quaternary structure. If it was a single
B. The protein could be an enzyme
protein and you see 4 bands, you can deduce that the single protein
was made of four subunits – and that is what you are seeing on the
C. The larger peptides traveled further than the smaller peptides
gel. (B makes no sense as you can’t determine what the protein
D. Restriction enzymes were used to create the fragments
39. Why is SDS used before protein electrophoresis? (2 pts)
SDS coats the protein in a negative charge and unfolds
the protein into its primary structure
does just from the gel; C is wrong, smaller fragments travel faster
than larger fragments and D is also incorrect – restriction enzymes
do not cut protein).
40. How does protein electrophoresis differ from DNA electrophoresis? (Choose all correct statements) (2 pts)
A. The DNA molecules are negatively charged, while the protein molecules during electrophoresis are positive
B. DNA electrophoresis is sorted in agar, while the protein electrophoresis is sorted with polyacrylamide gel
C. DNA fragments are sorted by size, while protein fragments are sorted by amino acids
D. DNA fragments are sorted by charge, while protein fragments are sorted by amino acids
41. With the help of your partner, you performed an SDS-PAGE. Unfortunately, your lab partner forgot to label what is
in each lane…. But we do know that Lane 2 is the size standard (it matches the size standard pattern given to us).
Neg (-), BLACK
A. Label the + and – ends of the gel. (0.5 pts)
B. Label the black and red ends of the gel. (0.5 pts)
C. Which lane contains cellular proteins after treatment of lysozyme, but
before being placed in the column? (2 pts)
Lane 1
D. Which lane contains the very last sample that was collected from the
column? (2 pts)
Lane 4
E. Measure the distance from the well to the center of the Lane 3 bands.
Band 1 = ___45 mm___ (1 pt)
Band 2 = ___52 mm __(1 pt)
Pos (+)
RED
F. Which protein is larger B or C? ___B __ (2 pts)
42. Approximately how large is a protein that migrated 34 mm? (2 pts)
Any number between 40-45 (1.5) – kilodaltons/kD/kDa (.5)
4.1.5: Protein Manufacturing
43. Protein production of insulin has been perfected so that it’s just like
following a recipe. What do you think could have been three problems
researchers had to overcome in order to perfect the technique? (2 pts)
Suggested answers could be: finding the appropriate restriction
enzymes, finding the best temperature for heat shocking or bacterial
growth, discovering ways of separating insulin (or other protein),
discovering ways of transforming bacteria or finding the best bacteria
for cloning.
44. One of the major goals of this unit was to think about the overall production and purification of a protein. First, give
the order of the steps necessary to produce and purify GFP. Second, give the importance of that step relating to the
goal of having a tube of pure protein product.
Process
SDS-PAGE gel
electrophoresis
Make
recombinant
DNA
Step #
4
1
Hydrophobic
3
Interaction
Chromatography
(HIC)
Bacterial
Transformation
2
Importance of step in the process of obtaining pure protein product.
This step simply verifies that your HIC fraction contains pure protein product. If you are
going to use this protein for commercial use or further research, you will want to make
sure you have pure product. The SDS denatures the proteins and coats them with negative
charges. The PAGE is a very tight matrix that will separate small molecules.
This step is important because you are making the plasmid with the gene of interest in it.
This plasmid is what will be inserted into the bacteria to produce the protein you want.
The plasmid helps the cell know what to do with the gene (i.e. promoters/terminators) and
has genes that help select for the cells that have taken in the new gene (i.e. GFP/amp
resistance)
In this step, you are isolating just the protein that you want. The protein is trapped in the
cell (with all of the other proteins the cell is making). You have to find a way to isolate the
protein of interest from the rest of the cell. In our case, the HIC step “grabs” the GFP
because of the hydrophobic nature of the protein. We can release the protein from the
column by changing the salt content and washing the protein from the column.
In this step, you are adding the recombinant plasmid to the bacteria. Some bacteria will
take in the new plasmid and start producing the new protein.
45. Which step is NOT specifically included in the steps above? Explain what occurs during the “missing” step. (2 points)
DNA Isolation – get the DNA from the cells then amplify the target gene with PCR
OR
Sales and Marketing – try to sell the drug to companies