Laboratory 11
Soil Enzyme Activity Part II
(Alkaline Phosphatase Assay)
Introduction
Bacteria and fungi that break down insoluble nutrient sources in the soil produce
extracellular enzymes. These are proteins that are produced inside the cell and exported out into
the soil solution. The enzymes are active outside the cell where they catalyze reactions to break
down the structure of the nutrient source to make it more accessible. The amount of an
extracellular enzyme in the soil depends on the metabolic abilities of the soil organisms, the
number of organisms present, the presence of substrate and the environment of the soil (pH,
temp., ionic strength etc.). Because enzymes are costly for the cells to make, they are tightly
regulated. Enzymes will only be made when they are needed.
One example of a common extracellular enzyme in soil is alkaline phosphatase. This
enzyme is produced by many organisms in the soil. Its purpose is to remove the phosphate
molecule from organic compounds such as phospholipids and nucleic acids. Once the phosphate
is cleaved it becomes soluble and can be taken up by the cell. This is a very important activity
because phosphate is often the limiting nutrient for microbial growth in soil.
In this lab you will be measuring the amount of active enzyme in soil samples by using a
chromogenic substrate assay. In the presence of alkaline phosphatase, the colorless chemical
para-nitrophenol phosphate is converted to para-nitrophenol, which is bright yellow. The
amount of product formed can be measured using a spectrophotometer and the amount of
enzyme activity can be calculated. You will also calculate the dry weight of the soil in order to
standardize the results. The soils that you will be analyzing have been kept moist and incubated
for ~2 weeks with the following amendments: 1.6g of yeast extract, 0.2g of inorganic fertilizer,
1.6g of yeast extract and 0.2g of inorganic fertilizer, or no addition.
Materials
Equipment
- incubator (37C)
- pH strips
- clinical centrifuge
- 5 ml pipettes and pumps
- screw-top tubes
- 13 X 100 mm test tubes
- balance
- spectrophotometer (460nm)
- drying oven (100C)
- aluminum weighing dishes
Samples
- one soil sample treated with organic
fertilizer, inorganic fertilizer, both or
untreated
Media and Reagents
- buffer (pH 10)
- 2 mM p-nitrophenol
- 0.5 M CaCl2
- PNPP test solution
(para-nitrophenol phosphate in buffer)
Procedures
Phosphatase Assay
1.
Weigh out two 2-gram portions of each soil sample and pour them into labeled screw-cap
tubes.
2.
Pipette 5ml of 0.5 M CaCl2 solution into each tube and shake well.
3.
Pipette 1ml of PNPP solution into one tube from each soil sample.
4.
Pipette 1ml of phosphate buffer into the other tube to serve as a control.
5.
Incubate all tubes at 37C for 1 hour.
6.
Centrifuge the screw-cap tubes in the clinical centrifuge for 5 min.
7.
Transfer 4ml of the supernatant into labeled 13 X 100mm test tubes.
8.
Centrifuge the test tubes in the clinical centrifuge for 5 min.
9.
Transfer 3ml of the supernatant into clean test tubes.
10.
Set the wavelength on the spectrophotometer to 460nm.
11.
Set the absorbance to zero with a blank tube containing 3 ml of CaCl2.
12.
Read and record the absorbance for each of your samples.
13.
Use pH strips to check the pH of each sample.
14.
Read and record the absorbance of the prepared standards.
15.
Plot the absorbance vs. concentration to make a standard curve.
Water Content Analysis
1.
Weigh four aluminum dishes (one for each soil) and record the weights.
2.
Weigh out ~10g of each soil sample in an aluminum dish. Record the exact weight.
3.
Put the samples in a 100C oven overnight and let them cool in a desicator.
4.
Weigh each of the dried samples and record the weight.
DATA Lab 11
20 points
Name:
Date:
Spectrophotometer Readings
Absorbance
Net Absorbance
(absorbance – control)
Concentration of
p-Nitrophenol
Organic fertilizer
control (organic)
Inorganic fertilizer
control (inorganic)
Combined ferilizer
Control (combined)
Unamended
control (unamended)
Standard Curve
Concentration
absorbance
2.0 mM
Water Content Analysis
Sample
Dish weight
Wet weight with dish
Wet weight – dish weight
Dry weight with dish
Dry weight – dish weight
Water content
1.0 mM
organic
0.5 mM
inorganic
0.25 mM
combined
Calculations
Water Content of Soil
Water content = (wet weight – dry weight) / dry weight
Dry weight = (wet weight / water content + 1)
0.125 mM
0.063 mM
unamended
Enzyme Activity
One unit of enzyme activity (U) is defined as the amount of enzyme that is able to
convert 1 mole of substrate to product in one minute. For soil assays, activity is
reported as U per gram of dry soil
1. Calculate the amount of p-nitrophenol that was produced using the standard curve
(remember that the total volume of liquid was 6ml but you only measured 3ml).
_______ µmoles in 6 mls
2. Divide the amount of product by the number of minutes that the samples were incubated
to find the value of U
_______ µmoles / minute
3. Calculate the dry weight of the soil sample that was used in the incubation.
_______ grams
4. Calculate the activity per gram of dry soil.
_______ U / gram of dry soil
Laboratory 12
DNA Part I
Extraction from Activated Sludge
Introduction
Many different techniques have been developed for extracting DNA from bacterial cells
and environmental samples. Some methods are very complicated while others are quite simple.
Your choice of technique depends on the specific sample that you are working with and your
requirements for the quality of DNA extracted. All of the methods include: a step for breaking
open (lysing) the cells to release the DNA, a step for removing all of the proteins and other cell
components, and a step to precipitate the purified DNA. In this lab you will be using a relatively
simple method. The cells are lysed by exposing them to repeated freeze / thaw cycles and a
detergent (SDS). The proteins etc. are extracted away by using phenol:chloroform, and the DNA
is precipitated with salt and alcohol.
The DNA extracted in this lab will be used to assess the diversity of microbes in different
activated sludge samples.
Materials
Equipment
- micropipettes
- microcentrifuge
- Dry-ice / ethanol bath
- Hot water bath (37C)
- vortexer
Supplies
- microcentrifuge tubes
- micropipette tips
-
500 mM EDTA
lysis buffer
lysozyme solution
10% SDS
ice
phenol:chloroform
3.0 M sodium acetate
100% ethanol
sterile water
Procedures
Sludge Extraction
1. Measure out approximately 0.5 ml of each sludge sample into a microcentrifuge tube.
2. Spin at 14,000 rpm for 1 min. then remove the supernatant with a pipette.
3. Re-suspend the pellet in 75l of 500 mM EDTA by vortexing vigorously.
4. Freeze each sample for 30 seconds in dry-ice / ethanol bath
5. Thaw in a 37C water bath.
6. Repeat freeze-thaw cycle 2 more times.
7. Add 300 l of lysozyme solution in lysis buffer (~4mg/ml) and mix.
8. Incubate at 37˚C for 15 min.
9. Add 50 l of 10% SDS followed quickly by 800 l of phenol-chloroform.
10. Vortex for 1 minute to form an emulsion.
11. Spin in a micro-centrifuge at maximum speed (~14,000 RPM) for 3 minutes.
12. Remove the top phase with a pipette, avoiding the lower phase and any solids. Add this top
phase to a new microcentrifuge tube with 800 l of phenol-chloroform.
13. Vortex for 1 minute and spin for 3 minutes.
14. Remove the top layer and add it to a new microcentrifuge tube.
15. Add 50 l of 3.0 M sodium acetate and 1000 l of 100% ethanol and chill on ice.
16. Spin in microcentrifuge at max speed for 15 minutes.
17. Carefully remove the supernatant and allow the pellet to dry completely.
18. Re-suspend the pellet in 50 l of sterile water.
19. Freeze the pellet until the next period.
Laboratory 12
DNA Part II
Electrophoresis and PCR
Introduction
The DNA extraction protocol that you used in part 1 of this lab should have resulted in
samples containing the mixed genomic DNA of all of the bacteria in the sludge that you used. In
order to visualize the DNA, and to measure its size, we will be carrying out a simple gel
electrophoresis. Electrophoresis separates DNA according to its size by drawing it through an
agarose gel using an electric field. DNA is negatively charged so it is attracted to the positive
electrode in the chamber. As it moves through the agarose gel, larger pieces of DNA will be
slowed down more than smaller pieces. The absolute size of the DNA fragments is estimated by
comparing them to known standards.
After we have confirmed that DNA is present we will perform a PCR amplification to
isolate and detect the individual 16S rRNA genes from the mixed genomic DNA. The 16S
rRNA gene codes for a part of the ribosome and is present in all bacteria and archaea.
Differences in the DNA sequence of this gene can be used to distinguish between different
phylogenetic groups. PCR works by using short pieces of DNA (primers) that are homologous
to parts of the sequence of interest to locate a specific gene. Once the primers bind to the target
sequences, the stretch of DNA between them is copied. This process is repeated through many
cycles, and results in an exponential increase in the numbers of copies of the targeted gene.
We will test the extracted DNA for the presence of specific forms of the 16S rRNA gene
that are diagnostic for different groups. The 16S rRNA gene codes for a part of the ribosome
and is present in all bacteria and archaea. Differences in the DNA sequence of this gene can be
used to distinguish between different phylogenetic groups.
Materials
Equipment
- thermal cycler
- gel electrophoresis power source
- electrophoresis rig
- micropipettes
- Polaroid camera
- UV light box
Supplies
- 1% agarose gel
- TAE buffer
- ethidium bromide(10 mg/ml)
- sterile water
- PCR kit
- Bacteria specific primers
- Archaea specific primers
- Planctomycete specific primers
- loading dye
- DNA size ladder
Procedures
Electrophoresis
1. Mix each sample with loading dye (2l dye with 10l sample) on a sheet of parafilm.
2. Place the 1% agarose gel into the electrophoresis box and cover with cold TAE buffer.
3. Load all 12l of each sample (including a size standard) into consecutive wells of the agarose
gel.
4. Place the cover on top of the electrophoresis box (check that the wires are attached to the
correct electrodes).
5. Set the power supply to run for 30 min. at 125V.
6. After the gel has run, remove it and transfer to the ethidium bromide-staining bath.
7. Stain for 10 min. then transfer the gel to the UV light box and take a picture.
PCR
1. Carefully label 3 PCR tubes for each sludge DNA, 1 for the bacterial primers (bac), 1 for
archaeal (arc) and 1 for planctomycete (pla).
2. Label 1 PCR tube for each primer set as a negative control.
3. Transfer 49l of the bac “master mix” into each bac tube.
4. Transfer 49l of the arc “master mix” into each arc tube.
5. Transfer 49l of the pla “master mix” into each pla tube.
6. Add 1l of each DNA sample to the appropriate PCR tube and 1l of sterile water to the
negative controls.
7. Load the samples into the thermal-cycler and start the program.
Standard protocol (for each 50l reaction)
40.75 l
1.0 l
1.0 l
1.0 l
5.0 l
0.25 l
1.0 l
PCR grade water
dNTP mix (10mM each)
5’ primer (~5 pmol/l)
3’ primer (~5 pmol/l)
10X NovaTaq buffer with MgCl2
(1.25 U) NovaTaq DNA polymerase
DNA template (~10 ng)
Temperature Cycles
Melting
Denaturing
Annealing
Extension
Final Extension
95C
94C
55C
72C
72C
5 min.
30 sec.
60 sec.
90 sec.
10 min.
TAE Buffer
4.84 g Tris Base
1.14 ml glacial acetic acid
2 ml 0.5 M EDTA
Agarose gel
1% agarose dissolved in 1X TAE
PCR Primers
Bacterial (bac) ~1363 Bases
27f
5′ AGA GTT TGA TCC TGG CTC AG 3′
1390r
5′ GTT TGA CGG GCG GTG TGT RCA A 3′
Archaeal (arc) ~633 Bases
A571f
5′ GCY TAA AGS RYC CGT AGC 3′
UA1204r
5′ TTM GGG GCA TRC KKA CCT 3′
Planctomycete (pla) ~1344 Bases
PLA46f
5’ GAC TTG CAT GCC TAA TCC 3’
1390r
5’ GTT TGA CGG GCG GTG TGT RCA A 3′
M = C or A; Y = C or T; K = G or T; R = A or G; S = G or C; W = A or T
Laboratory 12
DNA Part III
Electrophoresis
Introduction
The product of PCR amplification is a large number of copies of the sequence targeted by
the primers used. When the genomic DNA that is used as a template for PCR is from a mixture
of bacterial species, then the PCR product will consist of many different, but similar, sequences.
The length of the products are determined by the position of the primers, therefore, the success of
PCR reactions can be evaluated by using gel electrophoresis to look for specific-length products.
In this lab we are using three sets of primers to to amplify parts of the 16S gene from
three phylogenetic groups that may be present in activated sludge. Two of the primer sets (bac
and arc) will result in products approximately 1350 bases long. The other (pla) will give a 633
base product. If the organisms were present in your sludge sample, then we will see a DNA band
of the appropriate size on the agarose gel.
Materials
Equipment
- gel electrophoresis power source
- electrophoresis rig
- micropipettes
- Polaroid camera
- UV light box
- 37˚C water bath
Supplies
- 1% agarose gels
- ethidium bromide
- loading buffer
- DNA size ladder
- Sterile distilled water
- Mnl-I reaction mix
- 0.75M sodium acetate
Procedures
Electrophoresis
1. Mix a sample from each PCR product with loading dye (2l dye with 10l sample).
2. Place the agarose gel into the electrophoresis box and cover with cold TAE buffer.
3. Load all 12l of each sample (including a size standard) into consecutive wells of the agarose
gel.
4. Place the cover on top of the electrophoresis box (check that the wires are attached to the
correct electrodes).
5. Set the power supply to run for 30 min. at 100V.
6. After the gel has run, remove it and transfer to the ethidium bromide-staining bath.
7. Stain for 10 min. then transfer the gel to the UV light box and take a picture.
DATA
Name:
20 points
Date:
Attach labeled copies of gel photos for DNA extraction and PCR products.
Laboratory 13
Iron Cycle
Introduction
Iron is the fourth most abundant element in the earth’s crust (after oxygen, silicon, and
aluminum). The average iron content of soil, sediment, and rocks is about 5%. Most of the iron
in soils is present as iron oxides. In fact the typical soil colors (brown, red and yellow) are partly
due to various iron oxides. The black color that is common in anaerobic mud is caused by the
presence of reduced iron sulfides.
Like many other elements, iron is "cycled" between its oxidized and reduced forms by a
variety of different processes. Some of these processes are chemical while others are biological.
Iron oxides can be used in place of oxygen by some microorganisms forming ferrous iron
(Fe(II)) from ferric iron (Fe(III)) (iron reduction). Other microorganisms complete the iron cycle
by catalyzing the oxidation of Fe(II) to Fe(III). This cycle is illustrated in figure 1.
Bacterial cells are "powered" by capturing some of the energy released during oxidationreduction (redox) reactions, and the amount of energy available to them is directly related to the
electron potential of the redox reactions that they are able to carry out. Cells capture this energy
by shuttling electrons between the chemical being oxidized (electron donor) and the chemical
being reduced (electron acceptor) while keeping them physically separated. In this way bacteria
act as batteries and develop an electrical gradient (potential) that they use to do work (ATP
synthesis, transport, motility etc.). By mediating these electrochemical reactions, bacteria
modify their external environment such that it becomes more reduced.
The conditions that exist in stratified sediments (where highly reduced minerals (HS-,
Fe(II), NH4+ etc.) are produced by anaerobic metabolism deep in the sediment while oxygen is
present in the overlying water) form a natural electron gradient. The oxygen is prevented from
directly reacting with the reduced minerals because it is quickly used up by facultative aerobes at
the sediment surface. Recently it was realized that this natural electron gradient could be
converted into a fuel cell for harvesting electricity from the sea floor by embedding a graphite
electrode under the surface of the sediment and placing another in the overlying water (Reimers
et al., 2001). Electrons are transferred to the anode by diffusion of a number of reduced species
in the sediment including HS-, Fe(II) and humic acids. Interestingly, it has also been shown that
certain bacteria can directly transfer electrons from their cytochromes to the anode (Bond et al.,
2002).
In this lab we will be directly visualizing the generation of an electrical potential by the
activity of anaerobic bacteria. The potential that we measure is a reflection of the energy
available to the bacteria. We will then correlate the electrical activity to the changing reducediron gradient.
References
1. Reimers, C.E., L.M. Tender, S. Fertig and W. Wang. 2001. Harvesting Energy from the Marine SedimentWater Interface. Environ. Sci. Technol. 35:192-195.
2. Bond, D.R., D.E. Holmes, L.M. Tender and D.R. Lovley. 2002. Electrode-Reducing Microorganisms that
Harvest Energy from Marine Sediments. Science 295:483-485.
Figure 1. The Biological Iron Cycle
Fe(II) + O2
Fe(III) + H20 + energy
Fe (III)
IRON-OXIDIZING
BACTERIA
AEROBIC
Fe (II)
ANAEROBIC
IRON-REDUCING
BACTERIA
Fe(III) + organic matter
Fe(II) + CO2 + energy
Materials
Equipment
- Spectrophotometer (562 nm)
- balance
- voltage meter
Cultures
- lake sediment
Supplies
- iron-coated sand
- 1 liter clear water bottles
- silicone caulk
-
-
surface water
nutrient broth base
0.5 N HCl
ferrozine reagent (1 gram of
ferrozine (Sigma) per liter in 50 mM
HEPES buffer)
iron (II) standards (0, 0.2, 0.5, 1.25,
2.5 and 5.0 mM FeCl2 in 0.5 N HCl)
16 X 100 mm test tubes
pipette tips
syringes and needles
Procedures
Assembling the Column:
1. Put four "dabs" of silicone caulking approx. 1.5 inches apart on the side of the water bottle
(see the diagram). (the caulking needs to dry for 2 days before a sample can be taken through
it)
2. Punch a small hole next to the bottom sampling port and one next to the top port.
3. Insert a graphite electrode into each hole and seal with silicone caulk.
4. Measure ~150 ml of soil or sediment.
5. Weigh out 1.5 grams of nutrient broth base and mix with the sediment.
6. Pour the mixed slurry into the bottom of the water bottle, being careful not to have it stick to
the sides or cover the bottom electrode.
7. Fill the bottle to within 2-3 inches of the top with iron-coated sand. Stop filling just below
the top electrode.
8. Slowly fill the bottle to the top with pond water and leave uncapped.
9. Incubate at room temperature for five weeks.
Figure 2 The Iron Column
Colorimetric Assay:
1. Label four glass test tubes.
2. Remove 0.1 ml of water from each location on the column by using a syringe and needle.
3. Transfer each sample to the labeled test tubes and add 3 ml of 0.5N hydrochloric acid.
4. Add 6 ml of ferrozine reagent to each tube and to the standard curve.
5. Shake and let stand for 5 minutes.
6. Measure the absorbance (O.D.562) using a spectrophotometer.
7. Calculate the concentration of Fe(II) by using the standard curve.
Electric Potential:
1. Set the multimeter to read at the 2000mV range.
2. Attach the positive lead of the voltmeter to the top electrode and the negative lead to the
bottom electrode.
3. Read the meter to determine the voltage difference.
DATA Lab 13
10 points
Name:
Date:
Standards
Day 0
O.D.562
Concentration (mM)
Day 35
O.D.562
0.00
0.20
0.50
1.25
2.50
5.00
Samples
O.D.562
Day 0
Conc. (mM)
Day 35
O.D.562
Conc. (mM)
A (top)
B
C
D (bottom)
Day 0
Day 7
Day 14
Day 28
Day 35
Voltage
Question:
1). How could you change the conditions in the column so that more electricity was produced?