(Student Analysis Sheet)
Analysis Steps 1-4:
Teacher's notes:
Step 1. Measure the distance of the DNA bands (in mm) from each of the three
sample wells and draw an illustration of the DNA bands in each gel lane.
Note: The diagram of a gel on the left, has an example of a student's drawn
illustration of the DNA bands in one lane.
Step 2. Record migration distance and Rf values for each sample fragment imaged on the gel in the table below. Express fragment size in Kbp (Kilobase pairs)
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(1)
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Under a given set of electrophoretic conditions (i.e.,
pH, voltage, time, gel type, concentration, etc.) the
electrophoretic mobility of a DNA fragment molecule
is standard. Thus the length of a given DNA fragment
can be determined by comparing its electrophoretic
mobility on an agarose gel with that of a DNA marker
sample of known lengths. The smaller the DNA
fragment, the faster it will move down the gel during
electrophoresis. Each fragment has (under identical
electrophoretic conditions) a "relative mobility" value
(Rf). Rf can be expressed as:
distance the DNA fragment has migrated
from the origin (gel well)
distance from the origin to the reference
point (end of the gel)
A standard curve is constructed by plotting the Rf
value of each standard DNA fragment versus the
logarithm of its molecular size. The molecular size of
the DNA fragment is the antilog of this number. Both
standard curve plots and published fragment size data
are presented below.
Note: The values presented in the Experimental Data
Table on page 7 were obtained from the gel whose
image data was transferred to the drawing. Student
values will vary, however, they should be close to
published values (Table III) -at least to 2 significant
figures.
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(2)
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Standard curve plot(s) presented below. Note that
large DNA fragments are not on a line-of-best-fit.
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Step 3:
Compare your experimental data with the published data in the "Published Data
Table." Fragment base pair values within ( ) denote fragments too small to be
imaged on your gel. Your experimental values should agree with published data,
at least to 2 significant figures. Complete your table with published values
for fragments too small to be imaged.
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Step 4:
Use Table III to calculate cleavage site position order for each imaged and
non-imaged fragment. Write this value in the [ ] on your table. (Remember that
an RE that cuts linear DNA N times will yield N=1 fragments.)
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- Using the standard curve you constructed determine the molecular weight of the polypeptides assigned by your teacher.
- Answers will vary depending on the polypeptides (bands) chosen.
- Explain your rationale for using the gel banding pattern (DEF code) from each sample to classify and identify bacteria.
- Answer: Each imaged band represents one polypeptide; each polypeptide represents one gene, because genes are transcribed into messenger RNA and messenger RNA into polypeptides, it is possible to identify genetic relationships by comparing separated polypeptides imaged on a gel. To determine a perfect bacterial identification match, the two DEF codes must be identical. If the DEF codes are similar and only differ in several bands, then the two strains probably are closely related. The DEF code of distantly related or unrelated bacteria would be radically different.
- The enzyme lysozyme used to fractionate the bacterial cell wall is found in a great number of tissues and body fluids such as tears, blood and saliva. What do you suspect its function might be in these body fluids?
- Answer: This enzyme has antibiotic properties and along with other antibodies, serves as the first line of defense against harmful microorganisms. Lysozyme breaks down the peptidoglycan layer of bacterial cell walls, resulting in cell lysis. Antibiotics, such as penicillin, work in a similar fashion by inhibiting the cross-linking of peptide chains crucial for peptidoglycan synthesis.
- Why is the SDS solubilization buffer added after the bacteria cells are treated with lysozyme?
- Answer: SDS solubilizes the lipoproteins that make up the plasma membrane of the bacterial protoplast. This results in rupturing of the cell by inducing osmotic shock. The effect of the SDS solubilization buffer is minimal on bacterial cells whose bacterial cells have an intact peptidoglycan layer.
- Discuss the function of a reducing agent such as B-mercaptoethanol.
- Answer: A reducing agent cleaves the disulfide bonds that hold together the polypeptide constituents of whole proteins, making possible direct comparison of individual gene products, or polypeptides. For example, if a 125,000 Dalton untreated protein is electrophoresed it will yield one band. However, if the protein is treated with a reducing agent, it will yield several bands with a combined molecular weight of 125,000 Daltons-the same as the original protein.
- Examine your electrophoresis lanes. Based on the band patterns in these lanes, the background information in the introduction, and Figure 1, determine which samples are gram-positive and which are gram-negative. Explain the reasoning for your answers.
- Answer: Based on these electrophoresed samples, lanes 2 and 4 are gram-positive bacteria. The remaining lanes #1, 3 and 5 are gram-negative bacteria. Students should observe that certain samples show more banding patterns (overall) than others. Based on the information provided in Figure T1 as well as these sample results, students should conclude that gram-negative bacteria are chemically and structurally more complex than gram-positive bacteria. The electrophoretic patterns of gram-negative species reflect this overall structural complexity.
- Compare the banding pattern of Unknown A to the banding pattern of the three reference samples (E. coli, M. luteus, and S. marcescens D1) and identify it.
- Answer: Unknown A is Micrococcus luteus. Students should observe an exact correlation of protein patterns in lane 2 (the known standard) with lane 4 (the unknown). No other bacterium has this exact pattern. (Caution students to exercise care in their comparison--some variation in band definition will occur due to differences in applied sample volumes.) Thus electrophoresed unknowns can be compared with known strains to identify microorganisms without the use of gram-staining or morphological parameters. Utilizing electrophoresis to separate bacterial cell proteins, and comparing these separations to separations of reference strains stored in a computer database has important clinical applications. Students may notice that one unknown culture was the same color as one of the reference strains. Point out that other bacteria produce yellow pigments as well.
- Compare the banding pattern of Unknown B to the banding pattern of the three reference samples (E. coli, M. luteus, and S. marcescens D1) . Can you identify this unknown with certainty?
- Answer: The banding pattern of Unknown B (lane 5) bears a very close resemblance to that of Serratia marcescens D1 (lane 3). Students should note that this relationship is NOT a direct match. There are slight differences in the banding patterns, with Unknown B missing some bands that S. marcescens D1 possesses. Unknown B is Serratia marcescens WCF, the colorless mutant of S. marcescens D1. Strain WCF cannot produce a red cellular pigment (prodigiosin) present in strain D1. Students should conclude that strain WCF is missing certain gene sequences in its genome that code for this pigment. Some students may infer that Unknown B is E. coli on the basis of a lack of pigmentation. Such inferences are inconsistent with the banding patterns for both samples.
- In every sample lane, there is a distinct dark-staining band. Develop a hypothesis that explains what this band represents.
- Answer: The consistently dark-staining band in each sample, located at the bottom of the gel, is the protein lysozyme. Students should observe that this is the darkest band in every sample. Students should recall that this protein was consistently added in relatively large amounts (100 uL) during sample preparation. Some students also may suggest that its appearance at the bottom of the gel is consistent with its relatively low molecular mass--their supposition being that most enzymes have relatively low molecular masses. All of these factors are valid in forming their hypotheses.
References and Suggested Reading
Kreig, N.R., and J.G. Holt, eds. Bergey's Manual of Systematic Bacteriology, vol 1, 9th ed. Baltimore: Williams and Wilkins, 1984.
Starr, M.P. et al., eds. The Prokaryotes: A Handbook on Habitats. Isolation and Identification of Bacteria, 2 vols. Heidelberg: Springer-Verlag, 1981.
Volk, W.A., and M.F. Wheeler. Basic Microbiology, 5th ed. New York: Harper & Row, 1984.
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