Bioinformatic Analysis of Microbial Diversity:
Isolation, Amplification, Cloning and Sequence Analysis of 16S rRNA Sequences from Natural Microbial Communities
Bioinformatic analysis of nucleotide sequences of the small ribosomal subunit genes (16S and 18S rRNA genes) has become the method of choice to identify the microbes or microbial genera present in natural communities. These sequences are easily obtained from metagenomic DNA by amplification and cloning of 16S and 18S genes. Because approximately 99% of naturally occurring microbes cannot be cultured in the laboratory, bioinformatic analysis is the sole means of identification. An approximate timeline for these experiments is:
Day 1: Isolate metagenomic DNA from soil sample (0.5 hr), amplify 16S sequences by PCR (2.5 – 3 hr); ligate PCR products to the vector pCR2.1 (1 hr); transform competent DH5 cells with the ligation mixture (1.5 - 2 hr); and plate transformation mix to selective media to identify plasmid-bearing cells (0.5 hr)
Day 2: Inoculate cultures of transformants (0.5 hr)
Day 3: Isolate plasmid DNA containing 16S genes from these cultures ( 1 – 2 hr)
Day 4: Subject DNA to nucleotide sequence determination (24 hr)
Day 5: Bioinformatic analyses of 16S nucleotide sequences
The secondary structure of the 16S ribosomal RNA molecule, showing its single-stranded and base-paired regions. The 16S rRNA interacts with 21 proteins present in the 30S small ribosomal subunit through the numerous stem-loop structures shown in the diagram above. (Cover of Science 309, Sept., 2005.)
Laboratory 1: Isolation of Metagenomic DNA from Soil and Amplification of 16S rRNA Genes
Objectives of Laboratory 1A:
1. Isolate metagenomic DNA from a soil or sample
2. Subject eight reactions specific for the 16S gene of a microbial domain or genus to amplification by PCR
3. Prepare competent DH5 cells
Flow Chart of Laboratory 1A:
Isolate Metagenomic Set up Eight PCR ReactionsPrepare Competent
DNA from Soil Specific for 16S GenesDH5Cells
“It’s just astounding to see how constant, how conserved, certain sequence motifs—proteins, genes—have been over enormous expanses of time. You can see sequence patterns that have persisted probably for over three billion years. That’s far longer than mountain ranges last, than continents retain their shape.”
Carl Woese, 1997, In Perry and Staley, Microbiology.
INTRODUCTION: From the late 1800’s, when Koch cultured the anthrax bacillus and proved it was the causative agent of anthrax, until the mid-1980’s, scientists were confident they had identified most microbes present in the biosphere, estimated to include 107 to 109 different species of bacteria (Schloss and Handelsman, 2004). However, this identification was absolutely contingent on the ability to these microbes in the laboratory. Therefore, when new evidence gathered from aquatic and terrestrial ecosystems indicated that more than 99% of the microorganisms present in the environment could not be cultured in the laboratory and, thus, could be identified only by molecular means, shockwaves shook the scientific community to its core (Amann et al., 1995).
In 1977, Woese and Fox had proposed using ribosomal RNA (rRNA) gene sequences to classify bacteria and eukaryotes (Woese and Fox, 1977). The genes encoding the small ribosomal subunit (SSU, the 16S rRNA gene in bacteria and the 18S gene in eukaryotes; see Figure on next page) were selected because all species encode homologues of these genes and also because both the 16S and 18S genes contain both conserved and variable regions. Theapplication of this method, known as ribotyping, resulted in a re-classification of organisms into three kingdoms (Bacteria, Archaea, and Eucaryotes) rather than the five kingdoms that had been previously recognized. The advent of the Polymerase Chain Reaction greatly facilitated ribotyping, and The Ribosomal Database Project (Cole et al., 2005; was established for 16S and 18S sequences and is now in its second generation. RDP II curates over 101,600 16S rRNA gene sequences and includes both sequences amplified directly from the environment without prior culturing as well as sequences obtained from cultured microbes. Ribotyping has led to an enormous increase in the number of bacterial phyla, currently about 52, half of which are composed only of uncultured bacteria (Schloss and Handelsman, 2004; Rappe and Giovannoni, 2003). In fact, in July 2005, the number of 16S sequences from environmental organisms surpassed that from cultured organisms.
One method of studying uncultured microbes is to analyze genomic DNA isolated from a community of organisms. This type of study, in which DNA is obtained directly from the environment without prior culture, has spawned a new field known as metagenomics (Rondon et al. 2000; Handelsman. 2004). Metagenomics has not only facilitated analyses of genomic complexity and evolution but also resulted in the isolation of novel clones that express many different enzymatic activities, including anti-microbial compounds.
Today, you will first isolate metagenomic DNA from your soil sample and use this DNA to set up PCR reactions using primers specific for the 16S rRNA genes of either Bacteria or Archaea. Your reactions will be amplified over the noon hour, after which you will ligate the products of one PCR reaction to DNA of the vector pCR2.1. Later today, you will complete the process of cloning the amplified 16S rRNA genes from your soil sample by transforming the ligated products into E. coli. This procedure producesclones, which areexact copies.
Thepolymerase chain reaction (PCR) has revolutionized not only molecular biology but also numerous other scientific fields. PCR is a method by which a defined region of DNA is synthesized from minute amounts, even as little as a single DNA molecule, to yield quantities of DNA sufficient for detailed studies and analysis. This technique has become widely used in genetic diagnosis and forensics, as well as in innumerable basic research applications. The requirements for PCR include: a DNA polymerase to synthesize DNA, a DNA template for the polymerase to copy, the four deoxynucleoside triphosphates (dATP, dGTP, dCTP and dTTP) that are the building blocks of DNA, short DNA molecules (oligonucleotides) to serve as starting points or primers for DNA synthesis, and suitable reaction conditions for the DNA polymerase to synthesize DNA. PCR is usually performed using a thermally stable DNA polymerase known as Taq polymerase, which was isolated from Thermus aquaticus, a thermophilic bacterium that inhabits hot springs in Yellowstone National Park. In the reactions you will set up today, the template will be the DNA you isolated this morning from the microbes in your soil sample. The primers are short (15-25 bp) DNA molecules that function as starting sites for Taq polymerase to begin synthesizing DNA and are specific for the chromosomal region being amplified, in this case the 16S rRNA genes of soil microbes. The sequences of the primers are very important: they must be the exact complement (A pairing with T and G pairing with C) of sequences flanking the chromosomal region to be amplified.
The basic PCR cycle is composed of three steps or reactions, each of which is performed at a different temperature. In the first step, the template DNA is denatured at high temperature for a short time (94oC for 1 min in our reactions). In the second step, the temperature is lowered to allow the primers to anneal to the template DNA, again for a short time (20 sec at 43o C followed by 30 sec at 58oC). The 43o C incubation is necessary because some primers have low melting temperatures.In the third step, the temperature is raised to the optimal temperature for the DNA polymerase to synthesize DNA (72oC for 1 min). These steps are diagrammed in the Figure on the next page. Although the procedure is very rapid compared to many other techniques (a single three-reaction cycle usually requires less than four minutes), it is necessary to repeat this cycle thirty times to synthesize enough DNA for you to clone and also analyze by agarose gel electrophoresis.
In addition to being thermostable, Taq has the unusual characteristic of adding an extra “A” to the 3’ end of each sequence it amplifies. This additional base is very useful in the process of ligating the PCR products Taq produces to a plasmid vector. Without this additional “A”, the PCR products would have blunt ends, which ligate poorly. Consequently, far fewer ligation products will be produced. Plasmid vectors like pCR2.1 were created for the purpose of cloning PCR products by addition of a “T” to each of its 5’ endsto make these ends complementary to th e3’ ends of the PCR products.
II. EXPERIMENTAL PROCEDURES: Wear gloves and work only with your sample to avoid contaminating it with other microbes. Use cotton-plugged aerosol resistant tips (ARTips) at all times.
A. Processing Your Sample of Soil:1. Obtain a new Ziploc plastic bag and dump your soil sample from the tube you used to collect it into the bag. / / Label this bag with your initials and the date.
2. Composit (mix) your soil sample by inverting and massaging the bag several times. / / Don’t open the bag so the soil will remain sterile.
3. Although you will not sieve your soil sample today, sieving removes large particles and also helps mix the soil.
B. Isolation of Metagenomic DNA from Your Soil Sample: This procedure was delineated by MO BIO Laboratories, Inc., and is more rapid than comparable DNA isolation kits. / / You will use the Powersoil DNA Isolation Kit from MO BIO Laboratories, Inc. (#12888-50 or 12888-100)
1.Obtain an Isotherm and ice from near the large sink before beginning. Be sure to wear gloves and ask for help weighing your sample if needed. / / Spatulas can be sterilized by rinsing them in alcohol. Weighing instructions are next to the balances.
2. Use a sterile spatula and a small weigh boat to weigh out 250 mg (0.25 g) of soil. / / 0.25 g of damp soil is about the size of a large pea. Put the remainder of your soil into the cold box until tomorrow.
3. Add this 250 mg soil to a PowerBead Tube labeled with your initials. This tube contains small beads, which physically break cells open during the vortexing step.
4. Vortex gently to mix and disperse the soil. / / The PowerBead tube contains beads and a buffer to help disperse soil particles, dissolve humic acids and protect against DNA degradation. Humic acids can inhibit a variety of chemical reactions, including PCR.
5. Check that Solution C1 does not contain a precipitate. / / If a precipitate is present, heat this solution to 60o C until the precipitate is dissolved.
6. Add 60 l of solution C1 to the tube and invert several times to mix. / / Solution C1 contains the detergent SDS and other agents to completely lyse cells. SDS is an anionic detergent that disrupts lipids and fatty acids in cell membranes.
7. Secure your PowerBead Tube horizontally to a vortex mixer using the MO BIO vortex adapter tube holder or using tape to fasten it to a flat-bed vortex pad. / / If tape is used, check the apparatus often because tape can easily become loose.
8. Vortex at maximal speed for 10 minutes. / / This step is critical for complete lysis of the cells, which is caused by the chemical reagents in the PowerBead Tube as well as mechanical collisions between the beads and cells.
9. Place your PowerBead Tube into a microfuge. / / Make sure that the tube rotates freely in the microfuge without rubbing.
10. Centrifuge your tube at 10,000 x g for 30 sec at room temperature. (Conversion charts at Eppendorf URL) / / Do not exceed 10,000 x g or the tube may break.
11. Use your P200 to transfer the supernatant to a clean 2-ml collection tube that you have labeled with your initials. / / You should have 400 – 500 l supernatant, but the exact volume and color of the supernatant is unimportant.
12. Add 250 l of Solution C2 and vortex for 5 sec. / / Solution C2 will precipitate organic and non-organic material, including cell debris and protein.
13. Incubate at 4o C for 5 min.
14. Centrifuge your tube at rt (room temperature) for 1 min at 10,000 x g.
15. Avoiding the pellet, use your P200 to transfer up to 600 l of supernatant to a clean 2-ml collection tube. / / This is easy if you keep the pipet tip just below the meniscus of the supernatant.
16. Add 200 l of solution C3 to your tube and vortex briefly. / / This solution also precipitates organic and inorganic substances.
17. Incubate at 4o C for 5 min.
18. Again centrifuge your tube at rt for 1 min at 10,000 x g.
19. Again, avoid the pellet and use your P200 to transfer up to 750 l of supernatant to another clean 2-ml collection tube.
20. Add 1.2 ml of Solution C4 to the supernatant, being careful that the solution doesn’t overflow the rim of the tube. / / C4 contains a high concentration of salt, which will ensure that DNA binds tightly to the silica spin filters.
21. Mix very well by vortexing 5 sec and inverting tube several times.
22. Load approximately 675 l onto a spin filter and centrifuge at 10,000 x g for 1 min at rt.
23. Discard the flow through into a waste tube or beaker.
24. Add an additional 675 l supernatant onto the spin filter.
25. Centrifuge this tube at 10,000 x g for 1 min at rt again.
26. Again discard the flow through into the waste tube and load the remaining supernatant onto the spin filter. / / Three loads of supernatant are required.
27. Spin again at 10,000 x g for 1 min at rt and discard the flow through into the waste tube again. / / The DNA in your sample is now bound to the silica membrane in the spin filter.
28. Add 500 l of Solution C5 and centrifuge at rt for 30 sec at 10,000 x g. / / Solution C5 contains ethanol to wash contaminants from the precipitated DNA on the silica filter.
29. Discard the flow though from the 2 ml collection tube.
30. Centrifuge the spin filter at rt for 1 min at 10,000 x g. / / This spin removes residual solution C5.
31. Carefully place your spin filter into a clean 2 ml collection tube. / / Take care not to splash any solution C5 onto the spin filter.
32. Add 100 l of Solution C6 to the center of the white filter membrane. / / Solution C6 (10 mM Tris buffer) will elute the DNA from the spin filter. Placing this solution on the center of the filter will ensure that all areas are wetted.
33. Centrifuge at rt for 30 sec at 10,000 x g.
34. Discard the spin filter.
35. The metagenomic DNA you have just isolated is now ready for amplification by PCR as described next or for other applications. / The DNA should be kept on ice or stored frozen (-20o to -80o C) until ready for use.
You will now use this DNA to set up eight PCR reactions that arespecific for the 16S rRNA gene. / / Some reactions will amplify a genus or species-specific 16S rDNA, and others a universal rDNA.
One partner should follow the directions in Section C below to make an E. coli control for PCR, while the second partner should harvest DH5 cells as described in Section D below.
C. Making a Positive E. coli Control for PCR (Partner #1):
1. Obtain a clean 1.5-ml screw cap tube and add 50 l sterile water (clear tube, blue dot)to it. / / Label this tube with your initials and “K12” to denote E. coli K12. Keep the tube of water.
2. Obtain the stock plate of DH5 you worked with yesterday. / / DH5 is an E. coli K-12 strain.
3. Light your Bunsen burner with the striker.
4. Flame your loop until it glows red and touch it to the agar at the side of the plate away from any colonies. / / This cools the loop.
5. Use your loop to pick up a colony that is small to medium in size.
6. Transfer some E. coli cells to the water by moving the loop rapidly through the water in your tube.
7. Screw the cap onto your tube tightly.
8. Boil the water and E. coli for 5 min in a heating block. / / This lyses the cells, releasing their genomic DNA.
9. When the boiling step ends, put your tube in ice until you are ready to use it to set up a PCR reaction specific for E. coli. / / Preparing a sample like this control would be very easy to do in your classrooms.
D. Preparation of Competent DH5 Cells (Other Partner):Begin preparing competent DH5 following the instructions below. Be sure to use the plate that has been grown 5 days. / / The growth on this plate should be dense because the loop was not flamed after making the initial streak, and the streak made a tight zigzag pattern.
1. Obtain an LB plate onto which DH5 was streaked five days ago from the front bench as well as an orange-capped tube of LB broth. / / This plate was incubated at room temperature for 5 days and should contain rather dense growth.
2. Use your P1000 to add 1 ml LB broth from the orange-cappedtube to the surface of the plate and roll the plate to distribute the broth over as much of the surface as possible. / / Set your P1000 to “1-0-0”.
3. Locate the glass spreader (a glass rod bent into an “L” shape) at your bench and light your Bunsen burner with the striker. / / Place your burner in a spot where neither you nor your partner has to reach over it.
4. Sterilize your spreader by dipping it into the jar of ethanol at your bench and setting it aflame by putting it briefly into the flame of the burner.
5. The flame on the spreader will burn for only a couple of seconds before going out.
6. Lift the lid off the plate of DH5 and hold the lid above the plate to prevent contamination.
7. Briefly touch the spreader to an area of the plate that does not have any bacterial growth. / / This cools the spreader.
8. Move the spreader carefully across the entire surface of the plate to resuspend as many DH5 cells as possible in the L broth.
9. Tilt the plate at an angle by leaning it against a test tube rack to allow the broth to collect at the lowest point. Use your spreader to sweep broth into the puddle at the lowest point.
10. Use a sterile transfer pipet to transfer the resuspended cells to a 1.5 ml microtube. / / Label this tube with your initials.
11. Lay the plate flat on the bench and rinse the spreader with another 0.5 ml of LB while holding the spreader over the plate.
12. Move the spreader over the surface of the plate again to resuspend any remaining cells in the broth.