Exercise 6.

16S rRNA gene sequencing

6. Sequencing of the 16S rRNA gene.

BACKGROUND
Classification and determination of evolutionary relationship between bacteria have evolved quickly over the last two to three decades, where molecular techniques have been implemented worldwide in most microbiology laboratories. One of the key approaches was introduced by Carl Woese in the 1970s namely the use of the 16S rRNA gene (Woese and Fox, 1977). The gene encodes the ribosomal RNA that is a part of the small 30S subunit of the ribosome. Further two ribosomal RNA genes exist encoding the 5S rRNA and the 23S rRNA that are a part of the large 50S subunit of the ribosome. The advantage of the approach is the fact that the 16S rRNA gene is universally distributed among Bacteria and Archeaea and contain a good combination of conserved and variable regions allowing sequence alignment and detection of evolutionary relationships, respectively. Further, a continuously expanding number of sequences are becoming accessible through web-based database compilations (Maidak et al., 2001). The disadvantage is the level of identification possible, since the approx. 1600 bp of course only constitutes a small part of the complete genome, and essential differences can be hidden elsewhere. Therefore, for some parts of the phylogenetic tree, the separation can only be at the level of family or genus, whereas for other parts it can be down to species level. In no case, however, identification should be based solely on the 16S rRNA gene sequence. Analyses of the complete genome and/or physiological tests need to be included. Never the less, the 16S rRNA approach is straightforward and quick, - that is if you got access to the right equipment. PURPOSE The purpose of the exercise is to sequence the 16S rRNA gene from bacterial isolates cultured from gastrointestinal tract samples and to identify the isolates on this basis.

OUTLINE OF THE EXPERIMENT

Day 1 Step 1. Inoculation and growth of bacteria culture (1 hour + overnight). Day 2 Step 2. DNA extraction using bead beating and Geneclean kit (3 hours). Step 3. Start PCR with two primers to amplify the complete 16S rRNA gene (1 hour). Day 3 Step 4. Agarose gel electrophoresis. Purification of the PCR product using QIAquick kit (1 hours). Step 5. Start Cycle-PCR (1 hour + overnight). Day 4 Step 6. Precipitation of Cycle-PCR products (2 hours). Step 7. Separation of Cycle-PCR fragments on ABI sequenator (1 hour + overnight). Day 5 Step 8. Data-/sequence handling (4 hours). Bacterial isolates You start out by growing in batch culture cells from 20 single colonies from colon agar plates. You check the purity of the batch cultures by microscopy and extract DNA from 10 pure isolates. You PCR amplify the 16S rRNA gene of the 10 isolates and check the quality of the PCR products by agarose gel elctrophoresis. You sequence the 16S rRNA gene from 5 good quality PCR products (isolates) using three overlapping primers per isolates, each covering approx. 500 bp of the 1600 bp gene.

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Exercise 6. 16S rRNA gene sequencing

You end up with the following storage samples. 1. From step 2: 200-l samples of extracted genomic DNA (to be stored at -20C), from which 2 l samples are to be used for Step 3. 2. From step 4: 50-l samples of QIAquick-purified PCR-amplified 16S rDNA (to be stored at -20C), from which 1 l samples are to be used for Step 5. During the process you will have the following intermediary samples. 1. From step 3: 50-l samples of PCR-amplified 16S rDNA (can be stored at -20C). The whole sample has to be used in Step 4, Qiagen purification. 2. From step 6: Dry DNA pellet (can be stored at -20C). The whole sample has to be used in Step 7, gel separation. Step 1. Inoculation and incubation of overnight cultures (1 hour). Samples of faeces were taken from the rectum of the experimental animals and immediately transferred to a prereduced salt medium (See further Exercise 3). A dilution series was made and samples were spread plated on colon agar in an anaerobic cabinet (composition of atmosphere: 80% N2; 10% CO2; 10% H2). The plates were then incubated for 7 days in the cabinet. Team 1, 3 and, 5 should analyse colonies from the pigs fed coarse ground meal feed and Team 2, 4, and 6 should analyse colonies from the pigs fed fine ground pelleted feed. Important. The inoculation of the tubes is done in the anaerobic cabinet. 1. Each team should pick cells from 20 different and single colonies from the colon agar plates. 2. Use sterile toothpicks, inoculate 10 ml rumen-extract-glucose-cellulose medium in Hungate tubes with cells from the individual colonies and incubate at 37C for 24 h. Step 2. DNA extraction (3 hours, depending on number of samples) 1. Transfer a drop of bacteria suspension from each culture to a microscope slide and place a cover slip on top of the drop (you may have two samples on each slide). 2. Examine the cultures for purity using phase contrast or digital interference contrast (DIC or Numarski) microscopy. Describe the cell morphology (draw a sketch). 3. Harvest 1.0 ml bacteria cells from 10 pure cultures in Eppendorf tubes by centrifugation (13,000 rpm, 5 min). 4. Remove the supernatant and resuspend pellet in 1.0 ml TES buffer (0.05M Tris, 0.05M NaCl, 0.005M EDTA, pH 8.0). 5. Centrifuge (13,000 rpm, 5 min) and remove the supernatant. Pellet can be frozen at -20C to improve lysis of the cells. 6. Resuspend pellet in 500 l TES buffer. 7. Add 5 l lysozyme (10 mg per ml freshly prepared if possible), shake gently (i.e. turn the tube up and down once or twice) and incubate at 37C (thermo block) for 30 min. 8. Add 5 l Proteinase K (10 mg per ml) and 5 l RNase (10 mg per ml). Vortex and incubate at 65C (thermo block) for 1 hour. The increased temperature inhibits non-specific nucleases that would otherwise degrade DNA. 9. Add 50 l 24% SDS, vortex and incubate further 10 min at 65C. When the cells lyse the suspension will clear up. 10. While the lysis suspension cools down, add approx. 0.5 g glass beads (diam. 0.18 mm) to bead beat vials. 11. Pour the lysis suspension into the vial and bead-beat for 3 min. 12. Centrifuge (13.000 rpm, 10 min). 13. Prepare the Geneclean Turbo Purification columns by placing the filter unit in the collection tube. Write the sample name/number on the filter unit. 14. Transfer (use pipette) the supernatant (approx. 550 l) from the bead bet vial directly to the Geneclean column. 15. Close the filter lid and centrifuge the column (13.000 rpm, 1 min). 16. Separate filter and collection tube. Empty the collection tube (The DNA is bound to the filter matrix).

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Exercise 6. 16S rRNA gene sequencing

17. Sample filter and collection tube again and add 500 l wash-solution to the filter through the hole in the lid (do not open the lid). 18. Centrifuge the column (13.000 rpm, 1 min). 19. Separate filter and collection tube. Empty the collection tube. 20. Sample filter and collection tube again and centrifuge the empty column (13.000 rpm, 4 min) in order to dry the filter. 21. Remove the lid from the storage tube and place the dry filter in the storage tube. Write sample name/number on the storage tube. 22. Add 50 l eluation solution to the centre of the filter through the hole in the lid. 23. Leave the tube on the desk for 5 min. 24. Centrifuge (13.000 rpm, 1 min). 25. Remove the filter from the tube and close the storage tubes with the lid. 26. The extracted DNA is stored at -20C and should always be kept on ice when used on the bench. Step 3. PCR-amplification of 16S rDNA (1 hour + 2-3 hours run). Reaction master mix. Nucleotides dNTP (ATP, TTP, GTP, CTP) Primers (see sequences in appendix) 1 lb (S-D-Bact-0008-a-S-20) 6 lb (S-*-Univ-1510-a-A-19) DNA Polymerase DyNAzyme II (Finnzymes) PCR buffer from kit H2O (45 nmol per l) (5 pmol per l) (5 pmol per l) (2 U per l) (10) 2.0 l 2.0 l 2.0 l 0.5 l 5.0 l 36.5 l

DNA from Step 2 (Ideally 10 ng per ml) 2.0 l .. Total volume 50.0 l NB! Everything should be kept on ice until the PCR reaction is started. The master mix (minus DNA) is made up in a volume sufficient for all reactions and is dispersed with 48 l in each tube/well. The 2 l DNA can then be added. The PCR reaction can run in single tubes or in 96-well PCR plates. The reaction is performed on a Hybaid PCR Express thermo-cycler using the following program. Initial incubation Melting of DNA strands Annealing of primers Primer extension Fimnal extension Termination 95C: 60 sec 95C: 30 sec 60C: 30 sec 72C: 45 sec 72C: 10 min 4C: (ca. 2-3 hours) 30 cycles

The PCR-amplified samples of 16S rDNA can be stored at -20C, until purification is performed. It could be convenient to compile e.g. 96 samples from this step before purification and sequencing, so that these steps can be performed in 96-well plates, however it is not necessary (see Step 4).

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Exercise 6. 16S rRNA gene sequencing

Step 4. Agarose gel electrophoresis and purification of the PCR product with Qiagen purification kit, QIAquick (2 hours). Agarose gel elctrophoresis (control of DNA and PCR product). 1. Mix 100 ml TBE-buffer with 1 g agarose and boil the suspension in the microwave oven (max power, 2 min). 2. Let the solution cool a bit on the desk and add 5 l ethidium bromide solution (1%). Pour the solution into the electrophoresis chamber and place the comb in the solution. 3. When the gel is solidified, TBE is poured into the chamber and the comb is removed from the gel. 4. Gently mix samples of 2 l DNA with 0.5 l loading buffer on a piece of Parafilm. 5. Load 2 l of the samples into the lanes on the agarose gel. 6. Run the electrophoresis 4-5 min at 200 V. The blue indicator band should run approx. 0.5 cm into the gel. 7. Check the DNA bands on the UV table. Purification. The 50 l amplified DNA fragments from Step 3 is purified using a QIAquick PCR Purification Kit: Name QIAquick Single Spin Coloumns QIAquick 8-Well Strips QIAquick 96-Well Plates Cat. no. 28106 28144 28181 Number 2501 508 496 Price (DKK) 2.750,5.600,5.300,Price per sample 11,14,14,-

QIAquick Spin Coloumns are used together with a normal Eppendorf centrifuge, whereas QIAquick 8 strips and 96 plates demands a special vacuum unit or a microtiter plate rotor. In this experiment we will use Single Spin Columns and follow the commercial QIAquick protocol. QIAquick PCR purification kit protocol. 1. Add 250 l (5 volumes) Buffer PB to the 50 l (1 volume) PCR sample from step 3 and mix. 2. Place a QIAquick spin column in the 2 ml collection tube. Write sample name/number on top. 3. To bind the DNA, apply the PCR sample to column and centrifuge (13000 rpm, 1 min). DNA is now bound the column. 4. Discard flow-through solution from collection tube. Place the QIAquick column back into the same tube. 5. To wash the DNA, add 750 l Buffer PE to the QIAquick column and centrifuge (13000 rpm, 1 min). 6. Discard flow-through solution again and place the QIAquick column back into the same tube. 7. Centrifuge (13000 rpm, 1 min) the empty tube to remove residual ethanol/buffer from the column. 8. Place the QIAquick column in a clean 1.5 ml Eppendorf tube. Write sample name/number on tube. 9. To elute DNA, add 50 l Buffer EB (10 mM Tris-Cl, pH 8.5) to the centre of the QIAquick membrane and centrifuge (13000 rpm, 1 min). 10. The approx. 50 l QIAquick-purified 16S rDNA samples are stored at -20C. Samples of 1 l is used for the cycle-PCR/sequencing step (step 5). An agarose gel can be run at this point to check the DNA. However, it is not extremely necessary since the purification step normally does not remove DNA from the sample. Step 5. Cycle-PCR with ABI PRISM BIGDYE kit (2 hours + ON). For sequencing of both strands of the complete 16S rRNA gene (approx. 1500-1600 bp) six primers are needed. Thus complete sequencing of 16S rDNA takes six tubes/gel lanes per isolate/DNA sample. Partial sequencing can be performed using e.g. one primer alone or three primers.

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Exercise 6. 16S rRNA gene sequencing

Reaction mixture BigDye terminators* Rxn buffer** Primer*** Elga H2O

(kit) (5) (10 pmol per l)

1.0 l 1.0 l 2.0 l 2.0 l

Purified DNA from Step 4 ([?]) 1.0 l Total volume 7.0 l For partial sequencing with one primer make a volume of master mix (minus DNA) sufficient for all reactions and disperse 6 l in each tube. The 1 l DNA can then be added. For full sequencing keep track of the different primers used. For this purpose, prepare a sample sheet. This should be used when loading the ABI sequenator (Step 7). In the present exercise we use three primers per isolate, namely general primers 2 , 4 and 5 lb (see further appendix).
*

Perkin Elmer cat. no. 430 3152. Includes labelled and unlabelled dNTPs. Rxn buffer (5): 400 mM Tris-HCl, 10 mM MgCl2, pH 9.0 Primers (Purchased from DNA Technology, Aarhus, Denmark): Bifidobacterium primers: TH008-f LL409-f LL894-f TH504-r TH1037-r PHr

**

***

General primers: 1. S-D-Bact-0008-a-S-20 2. S-*-Univ-518-a-A-18 3. S-D-Bact-804-a-A-20 4. S-D-Bact-905-a-S-20 5. S-D-Bact-1054-a-A-20 6. S-*-Univ-1510-a-A-19 See sequences and positions in appendix. PCR-program (seq on the Hybaid PCR-machine): Melting Annealing Elongation 95C: 10 sec 50C: 5 sec 62C: 4 min

99 cycles (ca. 7 hours)

Termination 4C:

The cycle-PCR is run overnight followed next day directly by precipitation (Step 6) and run of sequence gel on the ABI sequenator (Step 7). Step 6. Precipitation of cycle-PCR product (2 hours). This procedure is performed directly in the PCR tubes (racks or 96-well plates) from Step 5. 1. The cycle-PCR samples (7 l) are precipitated by addition of 1 l 3M sodium acetate and 15 l 96% EtOH. 2. The samples are then incubated at -20C for approx. 15 min. 3. The samples are centrifuged (3000 rpm, 45 min). 4. The plate is placed upside-down on a napkin and then centrifuged also upside-down - briefly at max. 1000 rpm. 5. The DNA is rinsed by adding 50 l 70% EtOH. 6. Centrifuge (3000 rpm, 45 min).

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Exercise 6. 16S rRNA gene sequencing

7. The ethanol is poured off and the plate is again placed upside down on a napkin followed by centrifugation (upside-down) briefly at max. 1000 rpm. 8. Air-dry the pellet. 9. The dry pellet can be stored at -20C until run of the gel (Step 7), or you can continue directly with Step 7. Step 7. Separation of sequence fragments on ABI sequenator (1 hour + overnight). The following is a description of the procedure for preparing the sequence gel and setting up the ABI sequenator. The technician will do this step, but for your information we include it here anyway. 1. Wash glass plates (front plate (with cut and ground joint) and back plate). Be especially careful to clean the side with the ground joint (front plate) and the side with reversed writing (back plate), since these sides are facing the gel directly. Therefore use a) soap + hot water; b) deionised water from tap; c) Elga water. 3. Sampling of the plates. Place the back plate (no ground joint) in the bottom of the frame wiht the reversed writing upwards. Moisten the spacers (white plastic) and place them to follow the edge of the glass plates (the cut in the spacers should turn inwards and upwards). Place to front plate (ground joint downwards) on top of the other and tighten the clamps along the sides. Mount the gel-pulling aggregate. Check that it is neither wet nor dirty and that the hole for the syringe is clear. Mount the syringe (dry and clean) and place the plates in the fume hood. 4. Prepare an acrylic amide gel matrix by following the instructions on the package. The content is poured into a beaker glass and then directly into the syringe. Knock on the glass plate as the front of the gel advances to avoid air bobbles to get stocked in the gel. Mount the comb with the backside (no teeth) towards the gel and place clamps on the glass plate around it. The gel should now solidify for at least 1 hours. 5. The gel plates are now cleaned to remove acrylic amide. This will otherwise burn into the plates. Be careful to clean around the black cross pin, where the laser light from the reader will penetrate. The comb is removed and the well is rinsed with Elga water. The comb is cleaned in water and placed in the well with the teeth pressed ca. mm into the gel. 6. Place the bottom buffer chamber and the gel in the ABI. If the folder RA.MAIN4 is opened, close it and log the network connection off. In ABI Prism choose New Sequence Run, and run a plate-check (peaks = dirt on the glass plates where the laser light should penetrate. Clean the plates again and run another plate check. Meanwhile prepare 1400 ml TBE buffer. 7. When the plate-check is OK, place the upper buffer chamber in the ABI connect the wires. Fil the upper chamber with buffer (white edge of the comb) Check that the chamber is tight. Then fil the lower chamber. Mount the heating plate and connect power and water. Close the door. 8. Prerun. ABI Prism is started (Collection Sequence Run new.). Settings are checked (according to Anettes book). A prerun is started, where the temperature will increase to 51C. While the temperature stabilises, the sample sheet is opened (new Sequence Sample). The sample sheet is named and saved. Then prepare the samples. Loading of the sequence gel 1. Resuspend the DNA pellet from step 6 in 2 l loading buffer (800 l formamide + 200 l colour). 2. For denaturation of the DNA, heat the tubes (94C, 2-4 min) in the PCR machine. Transfer the tubes immediately thereafter to ice and keep them there until the samples are loaded on the gel. 3. Check that the temperature of the sequenator is 51C and pause it (press OK!). The sequence gel is loaded with 1-l samples in each lane using a multipipette. The samples are placed on the gel according to a sample sheet prepared in advance (see step 5). Every second (uneven) lane is loaded first. The machine is closed and started (press Resume), and runs for approx. 2 min. Pause again and load the even lanes. Then cancel the pre-run, install your sample sheet and start the run. Step 8. Data handling (4 hours). 1. The result of the sequence run (Run Folder) is saved in the Ole or the Lene folder under Sequences being analysed on the BioMol network.
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Exercise 6. 16S rRNA gene sequencing

2. The Run Folder is copied to the desktop. 3. The Run folder is opened. The gel picture (saved as xxx.seq) is opened in Gene Scan. The gel picture is checked visually and the tracking is evaluated. If the 96 lanes are not tracked correctly you have to perform a manual tracking - if possible - followed by a new extraction of the data. 4. In Sequencing Analysis the chromatograms are opened and checked visually. Bad ends are blunted. Partial sequencing of the 16S rRNA gene. Import the chromatograms into the Sequencher program. Sequencher can align and group the sequences according to similarity. The sequences are exported as PC-readable text files onto a disk key. The files are opened in word and the sequences are transferred (cut and past) to the Blast search machine (http://www.ncbi.nlm.nih.gov/BLAST/), connected to GenBank. The returned results are saved. Complete sequencing of the 16S rRNA gene. Assembling of the individual fragments from the different primers are done in the program Auto Assembler (to be demonstrated). The established consensus sequences are exported and analysed in the Blast system as described above. REFERENCES

Maidak, B.L., J.R. Cole, T.G. Lilburn, C.T. Parker, P.R. Saxman, R.J. Farris, G.M. Garrity, G.J. Olsen, T.M. Schmidt, and J.M. Tiedje. 2001. The RDP-II (Ribosomal Database Project). Nucl. Acids. Res. 29:173-174
Woese, C.R., and G.E. Fox. 1977. Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proc. Natl. Acad. Sci. USA. 74:5088-5090 REAGENTS AND EQUIPMENT Step 1. Media (agar plates, broth, tubes) according to Exercise 3 Step 2. Eppendorf centrifuge Eppendorf tubes Thermo block 37C, 60C Agarose gel electrophoresis system TES buffer (0.05M Tris, 0.05M NaCl, 0.005M EDTA, pH 8.0) Lysozyme, 10 mg per ml in H2O (-20C) Proteinase K, 10 mg per ml in H2O (-20C) RNAse, 10 mg per ml in H2O (-20C) SDS, 24% in H2O (RT) Pipettes and tips Step 3. PCR tubes dNTP (5 nmol) Primer (5 nmol) Polymerase from kit

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Exercise 6. 16S rRNA gene sequencing

PCR buffer from kit Elga water PCR-machine (PCR Express, Hybaid) Step 4. TBE-buffer (10.8 g Tris-base, 5.5 g boric acid, 40 ml 0.5M EDTA (pH 8.0) - usually a 10 concentrated solution is made, which is diluted 1:9 in Elga water before use) Agarose Loading buffer for agarose gel (30% glycerol, 0.25% xyanol, Elga H2O) UV-table QIAquick Turbo Purification Kit (columns, tubes, buffer) Centrifuge Eppendorf tubes Step 5. PCR plate (96 wells) Big Dye Terminator Kit Rxn-buffer, 5 (400 mM Tris-HCl, 10 mM MgCl2, pH 9.0) Primers Elga H2O PCR-machine Step 6. Sodium acetate 3M EtOH (96%, cold) EtOh (70%) Centrifuge for plates (refrigerated) Step 7. Loading buffer (from kit)

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