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Using DNA technology: to explore marine bacterial diversity in a Coastal Georgia Salt Marsh.

An important aspect of teaching biology is to expose students to the concept of biodiversity. For this purpose, bacteria are excellent examples. Prokaryotes were the first inhabitants on Earth, surviving and even thriving under very harsh conditions as new species continuously evolved. In fact, it is believed that there are more than 5 x 1030 prokaryotes living on Earth today (Whitman et al., 1998). Our current knowledge of these tiny organisms is very limited, and less than 1% of all bacterial species have been described (Horner-Devine et al., 2004). However, the prominent roles bacteria play in nature are not easy to overlook: Their functions range from providing essential nutrients to plants through nitrogen-fixation (such as for Rhizobium leguminosarum) to enhancement of nutrient absorption in animal intestines (such as for Escherichia coli). As a result, identifying unknown species of bacteria and extending our understanding of known ones are important tasks for 21st Century scientists.

Individual bacteria are too small to be seen or distinguished by the naked eye, or even by a microscope, making identification difficult. The traditional ways to identify bacterial species are based on their morphological, developmental, and nutritional characteristics, but these may be both inefficient and inaccurate. Microbiologists now take advantage of the rapid development in DNA technology, using the sequence of the small subunit ribosomal gene, or 16S rRNA gene, as a type of molecular fingerprint to classify bacteria (Johnson, 1984).

The advanced placement (AP) biology class at Cedar Shoals High School in Athens, Georgia, learned how to explore bacterial biodiversity using molecular fingerprinting. We collected marine water samples, isolated bacterial colonies, extracted DNA, amplified and sequenced the 16S rRNA genes, and then compared the sequences to an Internet database to reveal the identity of the isolates. The project began with a field trip to the salt marshes on Sapelo Island, a barrier island in coastal Georgia, and was completed at our high school and in a laboratory at the University of Georgia. Here we describe how the bacterial biodiversity exercise was carried out, and discuss options for source material for bacterial isolation and flexibility in scheduling the laboratory exercise modules. This laboratory has both educational and practical value for the students; it is appropriate for both AP biology and introductory college biology classes.

Methods

The laboratory is divided into three modules. In the first, basic ecology and microbiology techniques are used to collect a water sample from the environment and isolate bacterial colonies on solid growth medium in Petri dishes. In the second, molecular biology methods are used to extract genomic DNA from selected colonies, and amplify and sequence the 16S rRNA gene. In the third, bioinformatic tools are used to compare the sequences to those available in a national DNA database to obtain information about the identity and ecology of the isolates. Our class of 20 students was divided into groups of four. Each student isolated his/her own bacterial colony so that each group worked with four isolates (although not all were successfully taken through to sequencing). Class size for this exercise is flexible, but 24 or fewer students is recommended. The entire exercise takes about one month to complete, but time can elapse between the scheduling of each module.

Phase One: Isolating Bacterial Colonies

In our class, a seawater sample from a salt marsh on Sapelo Island, Georgia, was used for bacterial isolation. However, water, sediment, or soil samples can be obtained from virtually any natural environment as starting material for this exercise.

Collecting Water & Isolating Bacteria

Materials

* sterile collection bottle

* thermometer

* salinity meter (refractometer)

* 10 test tubes filled with 9 ml of sterile seawater for serial dilution

* 10 plastic Petri dishes filled with YTSS agar or other appropriate medium

(To make YTSS agar, mix 4 g yeast extract, 2.5 g peptone, 20 g sea salts, and 18 g agar into one liter of distilled [H.sub.2]O; autoclave and pour into Petri dishes.)

* 1-ml plastic pipets

* pipet pumps

* plastic plate spreader

* parafilm strips

Procedures

1. Measure the salinity and temperature of the water sample (to establish a record of the conditions under which the bacteria were living).

2. Collect about 500 ml of salt marsh water in a sterile bottle; each group should collect its own sample.

3. Use serial dilution technique to dilute the water sample. To do this, label 10 test tubes containing 9 ml sterile sea water as the [10.sup.-1] through [10.sup.-10] dilutions.

4. Inoculate 1 ml of the sample water to the tube labeled [10.sup.-1] using a sterile 1-ml plastic pipet, vortex.

5. Inoculate 1 ml from the [10.sup.-1] tube into the [10.sup.-2] tube using a new sterile pipet, vortex.

6. Continue transferring down the dilution series until all tubes are inoculated.

7. Label the Petri dishes as the [10.sup.-1] through [10.sup.-10] plates to correspond with the test tube series.

8. Use sterile plastic pipets to remove 0.5 ml from each test tube of diluted salt marsh water and deposit it on the corresponding plate. Quickly spread the liquid evenly with a plastic plate spreader. Shield the plate with the plastic cover to avoid contamination by air-borne bacteria.

9. Repeat the same process for the remaining nine tubes.

10. Wrap a strip of parafilm around all plates to prevent drying.

11. Incubate all plates at room temperature in the dark for three to four days in an incubator or cardboard box. Prepare additional YTSS agar plates during this period for plating selected colonies.

Selecting Colonies

Materials

* 10 plastic Petri dishes filled with YTSS agar or other appropriate medium

* plastic loops

* parafilm strips

Procedures

1. After the incubation period, select a single large, well separated colony from one of the plates with a sterile plastic loop. Streak onto a new agar plate in a zigzag pattern and label the plate bottom with a name for the isolate, the date, and the student's name. Each group member should select his/her own bacterial colony.

2. Repeat the process above until the desired number of colonies is selected. Wrap a strip of parafilm around each new plate and place back in the incubator or box.

3. The process of picking a well-separated colony from the most recent plate and streaking onto a new plate should be repeated several times for each isolate to ensure that pure colonies are obtained. Our class repeated it twice.

Phase Two: DNA Isolation, 16S rRNA Gene Amplification & Gel Electrophoresis

DNA Isolation

A simple technique is used to extract bacterial DNA in a boiling water bath. Centrifugation separates the DNA and other soluble components from cell wall debris. DNA extraction takes less than an hour.

Materials

* 500 [micro]l micropipettors

* plastic loops

* boiling water bath

* microcentrifuge

* microcentrifuge tubes

* vortex

* sterile distilled [H.sub.2]O

Procedures

1. With a plastic loop, remove a single well-separated colony from the agar plate and transfer it to a test tube containing 500 [micro]l sterile [H.sub.2]O. Vortex the tube vigorously for 1 minute to disperse cell clumps into the water.

2. Close the tube cap and boil in a water bath for 10 minutes. Clamp the test tubes caps closed to prevent them from opening during boiling.

3. Remove the tubes from the water bath and centrifuge at 13,000 rpm for 10 minutes.

4. Without disturbing the pellet at the bottom, use a micropipet to carefully remove the clear supernatant (containing DNA) to another sterile test tube.

5. The DNA solution can be stored at 4[degrees] C for up to one week or frozen at -20[degrees] C for longer-term storage.

Amplifying Bacterial 16S rRNA Gene Using the Polymerase Chain Reaction (PCR)

The 16S rRNA gene is present in all prokaryotes. The gene has highly-conserved regions (that is, nearly identical sequence in all organisms) which are good sites for PCR primers to bind; it also has variable regions that are unique to each species, providing a signature sequence for species identification. The primary function of 16S rRNA in the cell is to support the ribosome structure and align messenger RNA during translation. The gene is about 1500 bp long (Lewin, 2004). Setting up the PCR reactions and loading the thermal cycler takes about an hour. The thermal cycler run time is an additional three hours.

Materials

* 100 [micro]l micropipettors

* micocentrifuge tubes

* PCR thermal cycler

* PCR reaction beads (commercially-available beads that contain all necessary reagents for PCR)

* Primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1522R (5'-AAGGAGGTGATCCANCCRCA-3')

* sterile water

Procedures

1. Add 1 [micro]l of the bacterial DNA solution to a microcentrifuge tube containing a PCR reaction bead (the beads contain dNTPs--the four nucleotides, DNA polymerase, and a buffer), along with 22 [micro]l d[H.sub.2]O and 1 [micro]l each of the forward (27F) and reverse (1522R) primer.

2. Set up another tube with 1 [micro]l d[H.sub.2]O in place of DNA for a negative control; since no DNA is added to this tube, amplification should not occur. This checks for unwanted contamination.

3. Flick test tubes gently to mix and dissolve the PCR bead.

4. Place the test tubes into a PCR thermal cycler with the following heating cycles:

94[degrees] C for 1 minute--DNA double strands are separated to single strands.

55[degrees] C for 1 minute--primers attach to the complementary regions of the 16S rRNA gene.

72[degrees] C for 1 minute--the bases bind to their complementary sites as the DNA strands elongate.

5. One PCR thermal cycler contains enough space for all students' test tubes. The reaction runs 35 cycles.

6. Store the amplified DNA in a 4[degrees] C fridge (up to one week) until gel electrophoresis.

Agarose Gel Electrophoresis

An agarose gel is run to check for successful amplification of the 16S rRNA gene. When an electrical current is applied, negatively-charged DNA loaded into the gel migrates from the negative to the positive electrode at speeds that vary based on the molecule size. The DNA in the gel is then stained with ethidium bromide, which fluoresces under UV light. Students should be warned of the mutagenic nature of ethidium bromide. Only the samples that show one band of amplified DNA of the same length as the expected PCR product (~1500 bp), and whose control shows no band should be prepared for sequencing. Loading, running, and analyzing the gel takes about an hour.

Materials

* electrophoresis apparatus

* 1% agarose gel

* 500 ml Tris-acetate-EDTA (TAE) buffer

* loading dye

* ethidium bromide

* DNA marker

* 20 [micro]l micropipettor

* UV transilluminator

Procedures

1. Mix agarose and buffer in appropriate ratios to obtain a 1% gel (1 g/100 ml) and carefully heat the mixture in a microwave for about 45 seconds to melt the agarose. Stop the microwave about halfway through and, using gloves to protect against heat, swirl the flask before continuing. Add ethidium bromide (final concentration 0.5 [micro]g/ml) before pouring.

2. Pour the gel into an electrophoresis unit; insert combs at one end of the gel to produce wells and allow the gel to cool to room temperature.

3. Mix 7 [micro]l of the PCR products and 1 [micro]l of blue loading dye on a piece of parafilm or in a separate microcentrifuge tube. 18 [micro]l of the PCR product will be remaining in the tube and should be stored at 40 C.

4. Load the mixtures into each well; load 5 [micro]l of DNA marker into the last well (it will serve as a molecular size marker later).

5. Run the gel at 5 volts/cm for 20 minutes.

6. Observe the gel on a UV transilluminator with the plastic cover closed (to protect against the UV light). The fluorescent bands show the presence of an amplified 16S rRNA gene if the PCR was successful.

Sequencing 16S rRNA Gene PCR Product

The amplified DNA has to be cleaned to remove excess primers and polymerase before sequencing. We used a MoBio. UltraClean PCR Clean-up kit, which filters the DNA out with a solid-phase silica membrane and washes away undesired PCR reagents.

Materials

* MoBio Laboratories UltraClean PCR Clean-up Kit

* centrifuge

* 500 [micro]l micropipettors

* the stored 18 [micro]l of PCR product that was not run on the gel

Procedures

1. Follow the manufacturer's instructions for DNA cleanup on the successful PCR reactions.

2. Submit the cleaned PCR products to a sequencing center along with the 27F primer for sequencing.

Phase Three: Analysis of the Bacterial Gene Sequence with BLAST

GenBank[R] is the genetic sequence database of the National Institutes of Health (NIH) and contains approximately 62,000,000 sequence records (as of August 2006) (GenBank, 2006). Scientists all around the world deposit DNA sequences at GenBank, including those derived from viruses, bacteria, and complex eukaryotic organisms. Therefore, this database is the ideal place to compare our new sequences with previously- deposited sequences and identify our bacterial isolates.

The bacterial DNA sequences are "polished" before being analyzed at GenBank. In the computer lab, students trim off the beginning and end of the sequence to remove most of the Ns. These are unidentified nucleotides in the DNA sequence that are typically clustered at the beginning and end of the sequence. To compare the salt marsh bacterial 16S rRNA gene sequences to the ones in GenBank, students submit a query to the computer program BLAST at the National Center for Biotechnology Information (NCBI) Web site (http://www.ncbi.nlm.nih.gov/BLAST/). The Web site is self-explanatory and easy to use. Students enter the trimmed sequences into the query field, and the program will find the most similar sequences that have been deposited in GenBank. As a rule of thumb, if a student's sequence has [greater than or equal to] 97% similarity to a sequence deposited at the NCBI GenBank database, then it is a member of the same species. If the new sequence has [greater than or equal to] 95% similarity to a previous sequence, then it is a member of the same genus. The lower the match, the greater the chance that the bacterium has never been described before and may be new to science.

Results

Our class project resulted in 11 sequences representing a variety of Georgia salt marsh bacterial species (Table 1). We found strains from four major bacterial groups: the Alphaproteobacteria, the Gammaproteobacteria, the Firmicutes, Marine Bacterial Diversity 281 and the Actinobacteria (Figure 1). The first two taxa are particularly abundant in seawater and have been found to account for 30-50% of the cells that make up marine bacterial communities (Giovannoni & Rappi, 2000). The Alphaproteobacteria and Gammaproteobacteria (strains CS-2, CS-3, CS-5, CS-6, and CS-9 in Table 1) have closest relatives in salty environments from Korea and Brazil. Several other isolates we obtained belong to the phylum Firmicutes (strains CS-1, CS-7, CS-10, and CS-11 in Table 1). Their closest relatives in the GenBank database are from India and Spain. Firmicutes are often endospore-forming bacteria. The endospores allow cells to survive in a metabolically-inert state for many years, and are particularly good for withstanding harsh environmental conditions (Schaechter et al., 2006). The remaining two isolates we obtained from the salt marsh are members of the Actinobacteria (strains CS-8 and CS-12). Although our Actinobacteria strains were isolated from seawater, their closest relatives in GenBank were found in soil and sediment environments (Table 1).

[FIGURE 1 OMITTED]

Most of our isolates were similar enough to previously-described bacteria to be considered members of a known genus (approximately 95% sequence similarity or better; Table 1). The most unique isolate, however, was CS-12, whose sequence was only 94% similar to a sequence previously deposited in GenBank.

As our class went through the BLAST program results, we all wondered about the amazing roles these tiny microbes play in nature and the immense impact they have on the ecological processes on Earth. Bacteria are numerous and everywhere, in the oceans and coastal seas, in the soil and air, on the inside and outside of most animals, plants, fungi, and protists. They make life and prosperity on Earth possible for higher organisms.

Discussion

One of the most important goals of this laboratory is to familiarize students with modern DNA technology including DNA isolation, PCR, gel electrophoresis, and the use of online sequence databases. At the same time, the laboratory exercise gives students a unique opportunity to learn important biological principles with a hands-on approach. A student might read about DNA extraction or PCR countless times in a textbook, but this can be a less meaningful learning experience than actually carrying out these process to answer an important biological question. Reading textbooks cannot replace real-life experiences; the two are essential complements to each other in a successful educational process.

While our class focused on salt marsh bacteria, the samples we studied could have come from virtually anywhere, such as a freshwater pond, a forest soil, or even particles from the air. The immense biodiversity on Earth makes it very possible to learn about poorly-understood bacterial species no matter where the samples are from. Consequently, the project yields different results every time because of differences in the bacteria present in the starting sample. Students could subsequently submit their new sequences to GenBank, along with information about the environmental conditions when the sample was collected, the place and time it was found, and other variables they measured. In this case, the new sequence becomes part of the GenBank database and is available to scientists all over the world. Consequently, the results of this laboratory exercise could contribute to scientific understanding of the identity and distribution of bacteria on Earth. This participation in building the scientific knowledge base will encourage students to undertake future scientific adventures.

Our class was able to complete this project because of the availability of scientific instrumentation (a thermal cycler for PCR, electrophoresis apparatus, a sequencing facility) at the University of Georgia. Undoubtedly, this collaboration enhanced the AP biology curriculum and showed how forming partnerships with research laboratories can be a long-term solution to equipment limitations for high school AP classes. Projects like this one open students' eyes to a spectrum of new biological methods. In the event that molecular biology instrumentation is not available, however, Phases 1 and 3 of the exercise can be carried out as stand-alone projects. Phase 1 teaches basic microbiological techniques and allows students to isolate their own bacterial cultures. Phase 3 teaches bioinformatics techniques and allows students to learn about sequence databases and sequence matching programs such as BLAST. Example sequences can be downloaded by the teacher from the GenBank database and then used by the students for analysis in the computer laboratory.

The field trip component of this exercise gave students an opportunity to experience the excitement of biology directly, and gain a better understanding of the marine environment. Field trips constitute an essential part of biology education by generating interest and excitement on the part of the students. In combination with the laboratory procedures, this exercise is a complete research process, in which students move from live organisms in their environment to their DNA, and from their DNA to biodiversity. The laboratory exercise also inspires critical thinking in students and can lead to hypothesis-driven follow-up experiments. For example, the 16S rRNA gene sequence of a marsh bacterium may be closest to that of a bacterium isolated earlier from a warm lagoon in Africa. It may be hypothesized that the newly-discovered bacterium can withstand high temperatures like its relative, and an experiment could be set up to test the hypothesis.

As time progresses, progress in science relies more and more on the availability of online resources. The inclusion of an online database activity in this exercise reflects the important relationship between computers, the Internet, and the development of biotechnology and biological knowledge. Bioinformatics can greatly reduce the workload of a researcher, allowing more time to be spent on non-repetitive work, and making valuable scientific information equally available to scientists all around the world. In this laboratory exercise, we easily obtained up-to-date information about the identity and distribution of bacterial species without even leaving our desk.

Acknowledgments

We would like to thank the National Science Foundation and the Gordon and Betty Moore Foundation, who kindly provided the funding for the field trip to Sapelo Island and the laboratory materials. We appreciate editing assistance from Sam Fahmy and data analysis assistance from Erinn Howard, both of whom are at the University of Georgia.

References

GenBank Overview. (2006). Available online at: http://www.ncbi.nlm.nih.gov/Genbank/.

Giovannoni, S. J. & Rappi, M. (2000). Evolution, diversity, and molecular ecology of marine prokaryotes. In D. L. Kirchman (Ed.), Microbial Ecology of the Oceans. NY: Wiley-Liss.

Horner-Devine, C. M., Carney, K. M. & Bohannan, B. J. M. (2004). An ecological perspective on bacterial biodiversity. Proceedings of the Royal Society B: Biological Science, 271, 113-122.

Johnson J. L. (1984). In N. R. Kreig & J. G. Holt (Eds.), Bergey's Manual of Systematic Bacteriology, 1, 8-11. Baltimore: Williams.

Lewin, B. (2004). Genes VIII. Saddle River, NJ: Pearson Education.

Schaechter, M., Ingraham, J. L. & Neidhardt, F. C. (2006). Microbe. Washington DC: ASM Press.

Whitman, W. B., Coleman, D. C. & Wiebe, W. J. (1998). Prokaryotes: The unseen majority. Proceedings of the National Academy of Sciences, 95(12), 6578-6583.

YIHE DONG (yihedong@gmail.com) is an AP biology student and STELLA GUERRERO (guerreros@clarke.k12.ga.us) is her AP biology teacher at Cedar Shoals High School, Athens, GA 30605. MARY ANN MORAN, Ph.D. (mmoran@uga.edu) is Professor at the University of Georgia, Marine Sciences Department, Athens, GA 30602.
Table 1. Taxonomy and ecology of the closest relatives to the Cedar
Shoals marine bacterial isolates based on comparisons of 16s rRNA gene
sequences using the BlAsT tool at the National Center for Biotechnology
information (NCBI) database. The "% similarity" column indicates the
percent of nucleotides with exact matches between the Cedar shoals
marine isolate sequence and the closest sequence in the NCBi database.
As a rule of thumb, a similarity of [greater than or equal to] 95%
indicates that two bacteria are members of the same genus.

Strain % Phylum/ Genus
Name Similarity Subphylum

CS-1 99 Firmicutes Exiguobacterium

CS-2 98 Gammaproteobacteria Microbulbifer

CS-3 99 Alphaproteobacteria Phaeobacter

CS-5 96 Gammaproteobacteria Salinimonas

CS-6 97 Alphaproteobacteria

CS-7 98 Firmicutes Bacillus

CS-8 94 Actinobacteria Arthrobacter
CS-9 98 Gammaproteobacteria Pantoea soil

CS-10 98 Firmicutes Exiguobcterium

CS-11 98 Firmicutes Bacillus

CS-12 94 Actinobacteria

Strain Location Ecology
Name

CS-1 Western psychrotrophic (cold-tolerant) and
 Himalayas, alkali-philic (alkaline-loving)
 India

CS-2 salt marsh, Korea halophilic (salt-loving)

CS-3 tidal flat, Yellow Sea,
 Korea

CS-5 solar saltern, Korea halophilic (salt-loving)

CS-6 mangrove sediments,
 Brazil

CS-7 arsenic contaminated
 aquifer, Bengal
 Delta, India

CS-8 soil, Saitama, Japan

CS-9 soil 2,4,6-trinitrotoluene (TNT)
 degrading

CS-10 Western Himalayas, India psychrotrophic (cold-tolerant) and
 alkali-philic (alkaline-loving)

CS-11 Cadiz, Spain intestine microflora of marine fish

CS-12 sediments, Taihu lake,
 China
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Title Annotation:INQUIRY & INVESTIGATION; deoxyribonucleic acid
Author:Dong, Yihe; Guerrero, Stella; Moran, Mary Ann
Publication:The American Biology Teacher
Article Type:Report
Geographic Code:1USA
Date:May 1, 2008
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