Laboratory 7: Analysis of Microbes from water and Soil ...



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

[pic]

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 Reactions Prepare Competent

DNA from Soil Specific for 16S Genes DH5( Cells

“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. The application 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 produces clones, which are exact copies.

The polymerase 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 (94o C 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 58o C). 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 (72o C 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.

[pic]

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’ ends to 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 |( |Label this bag with your initials and the |

|collect it into the bag. | |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 |( |You will use the Powersoil DNA Isolation |

|BIO Laboratories, Inc., and is more rapid than comparable DNA isolation kits. | |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 |( |Spatulas can be sterilized by rinsing them|

|gloves and ask for help weighing your sample if needed. | |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 |( |The PowerBead tube contains beads and a |

|small beads, which physically break cells open during the vortexing step. | |buffer to help disperse soil particles, |

| | |dissolve humic acids and protect against |

|4. Vortex gently to mix and disperse the soil. | |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 |( |If tape is used, check the apparatus often|

|tube holder or using tape to fasten it to a flat-bed vortex pad. | |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 |( |Do not exceed 10,000 x g or the tube may |

|Eppendorf URL) | |break. |

|11. Use your P200 to transfer the supernatant to a clean 2-ml collection tube that you have |( |You should have 400 – 500 (l supernatant, |

|labeled with your initials. | |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 |( |This is easy if you keep the pipet tip |

|2-ml collection tube. | |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 |( |C4 contains a high concentration of salt, |

|overflow the rim of the tube. | |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 |( |Three loads of supernatant are required. |

|onto the spin filter. | | |

|27. Spin again at 10,000 x g for 1 min at rt and discard the flow through into the waste tube|( |The DNA in your sample is now bound to the|

|again. | |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 | |The DNA should be kept on ice or stored |

|described next or for other applications. | |frozen (-20o to -80o C) until ready for |

| | |use. |

|You will now use this DNA to set up eight PCR reactions that are specific for the 16S rRNA |( |Some reactions will amplify a genus or |

|gene. | |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|( |Label this tube with your initials and |

|it. | |“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 |( |This cools the loop. |

|from any colonies. | | |

|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|( |Preparing a sample like this control would|

|PCR reaction specific for E. coli. | |be very easy to do in your classrooms. |

|D. Preparation of Competent DH5( Cells (Other Partner): Begin preparing competent DH5( |( |The growth on this plate should be dense |

|following the instructions below. Be sure to use the plate that has been grown 5 days. | |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|( |This plate was incubated at room |

|as an orange-capped tube of LB broth. | |temperature for 5 days and should contain|

| | |rather dense growth. |

|2. Use your P1000 to add 1 ml LB broth from the orange-capped tube to the surface of the |( |Set your P1000 to “1-0-0”. |

|plate and roll the plate to distribute the broth over as much of the surface as possible. | | |

|3. Locate the glass spreader (a glass rod bent into an “L” shape) at your bench and light |( |Place your burner in a spot where neither|

|your Bunsen burner with the striker. | |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 |( |This cools the spreader. |

|growth. | | |

|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. | | |

|13. Again lean the plate against a rack and allow the broth to collect at the lowest point. |( |Again use the spreader to sweep liquid |

| | |into the puddle. |

|14. Use another sterile transfer pipet to transfer this second aliquot of resuspended cells |( |Flick this tube to mix the contents. |

|to the same 1.5 ml tube. | | |

|15. Let the cells sit undisturbed at rt for exactly 2 hr 15 min over lunch. |( |This relatively long incubation produces |

| | |more competent cells. |

|16. Before proceeding, turn off your burner and wipe down your bench with 70% ethanol from a | | |

|wash bottle. Continue to prepare cells after lunch. | | |

|E. Setting up PCR Reactions for 16S rRNA Genes: Each lab pair will cooperate to set up seven |( |Continue to use ARTips when pipetting in |

|PCR reactions specific for the 16S rRNA genes of different microbes using the DNA you and | |order to prevent contamination. Additional|

|your partner just isolated from soil. The eighth reaction will be a positive control using | |racks are available if needed. |

|the E. coli DNA you just made. Both partners should cooperate to make these eight PCR | | |

|reactions. |( |Please change your gloves now before |

| | |beginning. |

|1. The first partner should take an Isotherm and ice to obtain seven tubes containing the |( |The primer mixtures contain equal parts |

|following primer mixtures from the front bench. | |of forward and reverse primers. |

|a. Universal primers for bacteria (0.65 ml tube labeled “FR”) |( |Biological Procedures Online. |

| | |bpo/arts/1/6/|

| | |m6.htm |

|b. Universal primers for archaea (0.65 ml tube labeled “A1”) |( |Baker et al. 2003. J. Microbiol. Meth. 55:|

| | |541. |

|c. Primers for Pseudomonas (0.65 ml tube labeled “Pseu”) |( |Milling et al. 2004. Plant & Soil 266: 23.|

|d. Primers for fungi, protists, and green algae (0.65 ml tube labeled “NS”) |( |White et al. In PCR Protocols, A Guide to |

| | |Methods and Applications. P. 315. |

|e. Primers for high G+C gram + bacteria, which includes Actinomyces (0.65 ml tube labeled |( |Kuske et al. 1998. Appl. Environ. |

|“Act”) | |Microbiol. 64: 2463. |

|f. Primers for Bacilli (0.65 ml tube labeled “Bac”) |( |Kuske et al. 1998. Appl. Environ. |

| | |Microbiol. 64: 2463. |

|g. Primers for E. coli K12 (0.65 ml tube labeled “E”). You will use these primers to set up |( |Epicentre Biotechnologies ()|

|two PCR reactions. | | |

|2. Obtain eight clear 0.5 ml Ready-To-Go PCR Bead Tubes from the front bench. |( |Each Ready-To-Go PCR Bead contains Taq |

| | |polymerase, the four dNTP's, MgCl2, KCl2, |

|3. Tap each tube gently on your bench to ensure the bead is at the bottom of the tube before | |and Tris-HCl buffer. |

|opening that tube. | | |

|4. Label the tops and sides of seven of these tubes using a fine-tipped black marker with one|( |Label the eighth tube “E-C” for “E. coli |

|of the seven letters/acronyms to designate the different primers, e.g., “A1”, “FR”, etc. | |control”. |

|5. Using your P20 set to “2-0-0” with a fresh 20 (l ARTip, add 20 (l of each of the primer |( |For example, add 20 (l “A1” primers to the|

|mixtures to the appropriately labeled Ready-To-Go Bead Tubes and the tube labeled “E-C”. Be | |tube labeled “A1”. Each mixture contains |

|sure to use a clean tip for each primer mixture. | |0.5 (l each of forward and reverse primers|

| | |(10 (M) and 19 (l H2O. |

|6. Flick all eight tubes gently but well with your fingers until the PCR beads are thoroughly|( |Keep these Ready-To-Go tubes cold in ice |

|dissolved. | |as often as possible. |

|7. Move the tube labeled “E-C” away from the other seven tubes. |( |You will add a different DNA template to |

| | |the “E-C” tube. |

|8. Using your P20 with a fresh 20 (l ARTip, transfer 5.0 (l of your soil DNA to each of the |( |Do not add soil DNA to the tube labeled |

|seven Ready-To-Go PCR Bead tubes you just prepared. | |“E-C”. |

|9. Flick these tubes gently but thoroughly to mix. | | |

|10. Transfer 5.0 (l of the E. coli DNA you prepared earlier to the tube labeled “E-C”. | | |

|11. Flick the “E-C” tube to mix the contents and spin all eight tubes 10 sec in the |( |This brings all droplets to the bottom of |

|microfuge. | |each tube. |

|12. One partner should obtain one strip of eight 0.2 ml PCR tubes and a PCR rack from the |( |Be sure to wear gloves when obtaining |

|front bench. (Omit steps 12- 14 if your cycler accommodates 0.5 ml tubes.) | |these tubes. |

|13. Use a black marker to label each PCR tube with at least one of your initials and one of |( |Label the top and hinge of each tube if |

|the seven acronyms (and another “E-C”) denoting the different primer pairs. | |possible. |

|14. Set your P200 to “0-3-5” (the bead adds volume) and transfer the entire contents of each |( |Transfer the contents of the “E-C” tube |

|Ready-To-Go Bead tube into the PCR tube with the matching label. | |last. |

|15. Return the tube containing the soil DNA you isolated to the front of the lab for storage.|( |Make sure this tube is clearly marked with|

| | |your initials and “Soil DNA”. |

|Bring your strip of eight PCR tubes to the front of the lab and put them into the PCR rack. |( |Your tubes will be put into an automated |

| | |thermal cycler that has been programmed |

| | |for the following conditions. |

|94o C 3 min | | |

|30 cycles: | | |

|Denaturing time and temperature: 1 min at 94o C | | |

|Annealing time and temperature: 20 sec at 43o C followed by 30 sec at 58o C |( |Annealing at 43o C is necessary because |

| | |some primers have low melting |

| | |temperatures. |

|Extending time and temperature: 1 min at 72o C | | |

|72o C 10 min | | |

Laboratory: Analysis and Cloning of 16S PCR Products

Objectives of Laboratory 1B:

1.Set up a ligation reaction using your “FR” PCR products and the vector pCR2.1

2. Analyze your PCR products using e-gel electrophoresis

3. Finish preparing competent DH5( cells

4. Transform your ligation reaction into these DH5( cells

5. Plate transformants onto selective plates

Flow Chart of Laboratory 1B:

Set up a Analyze PCR Products Transform DH5( Plate to Select

Ligation using Electrophoresis with Ligation Mix Transformants

I. INTRODUCTION: This morning you isolated metagenomic DNA from soil and set up several PCR reactions to amplify 16S sequences from this DNA. You will now continue your experiment by setting up a ligation reaction to ligate or tie the PCR products in your “FR” reaction to the plasmid vector, pCR2.1. This process is called cloning because clones are exact copies. Cloning has three important components or ingredients: 1) fragments of DNA to be cloned (your PCR products in today’s experiment); 2) a vector (an engineered plasmid or virus) into which the cloned fragments are inserted (pCR2.1 in your experiment); and 3) the enzyme DNA ligase that forms phosphodiester bonds between the DNA fragments and the vector DNA.

The vector pCR2.1 has several features that are essential for cloning: a replication origin which enables it to replicate independently of the host bacterial chromosome, genes for resistance to ampicillin (Ampr) and kanamycin (Kanr) which are used to select for cells that contain pCR2.1, a region known as a polylinker which contains cloning sites for 14 restriction enzymes, and a short segment (the LacZ ( fragment), which contains the regulatory sequences and coding information for 146 amino acids of the E. coli (-galactosidase (LacZ) gene. (-galactosidase is an enzyme that breaks down lactose into glucose and galactose. The polypeptide encoded by this region of LacZ is known as the ( subunit of (-galactosidase and is the basis for an easy assay that will allow you to determine visually whether a foreign DNA fragment has been inserted into the polylinker of pCR2.1. A diagram of pCR2.1 is shown on the next page.

In addition to being thermostable and able to remain active despite the elevated temperatures used for PCR, Taq has another advantage when used for PCR. Specifically, Taq has the unusual characteristic of adding an extra “A” to the 3’ ends of each DNA sequence it synthesizes. These additional bases are very useful in ligating PCR products to a vector because without these additional “A’s”, far fewer ligation products would be produced. Plasmid vectors like pCR2.1 that are used for cloning PCR products have been modified by addition of “T’s” to make the 3’ ends of the vector molecules complementary to the ends of the PCR products.

After you set up your ligation reaction, you will analyze all your PCR products using agarose gel electrophoresis. Then you will transform your ligation mix into competent DH5( cells you will prepare. Later today, you will plate your transformation mix onto special plates to select for cells that have taken up a plasmid.

[pic]

The diagram of pCR2.1 above illustrates several features of the vector that are essential in cloning, including 14 sites for restriction enzyme cleavage that flank the cloning site into which an exogenous DNA fragment can be inserted.

II. EXPERIMENTAL PROCEDURES: One partner should continue preparing competent DH5( cells (following the instructions in Section A below) while the second partner sets up a ligation reaction (as directed in Section B below):

|A. Partner #1- Preparation of Competent DH5( Cells: | | |

|1. Obtain fresh ice in your Isotherm and collect small tubes containing sterile solutions|( |Keep these tubes on ice. Calcium ions make |

|of 10, 30 and 100 mM CaCl2 from the front bench. | |bacterial cells permeable so the cells take |

| | |up DNA. |

|2. When the DH5( cells you harvested earlier have incubated at rt for 2 hr and 15 min, |( |Pour the LB broth supernatant carefully into |

|spin the cells at 5 K rpm for 1min and pour off the supe carefully. | |the waste container on your bench. |

|3. Add 1.0 ml 10 mM CaCl2 to the cells and flick the tube to mix the contents well. |( |Cells become quite fragile when treated with |

| | |CaCl2, so never vortex these cells. |

|4. Spin cells at 5 K rpm for 1 min and carefully pour off the supernatant. |( |Watch the pellet as you pour and discard the |

| | |supe into the waste container on your bench. |

|5. Add 0.3 ml of cold 30 mM CaCl2 to the cells and flick the tube to mix the contents |( |If the pellet does not resuspend well, use |

|well. | |your P200 set to 1-5-0 and a tip to squirt |

| | |CaCl2 at the pellet to resuspend the cells. |

|6. Leave these cells on ice for 10 – 20 min. | | |

|7. Spin cells at 5 K rpm for1 min again and pour off supe. |( |Watch the pellet as you pour. |

|8. Flick the tube with your fingers to partially resuspend the cells. |( |If the pellet is difficult to resuspend, use |

| | |your P200 again to resuspend the cells. |

|9. Add 0.3 ml cold 100 mM CaCl2 and use your P200 to resuspend the cells thoroughly. |( |Adjust the volume of 100 mM CaCl2 depending |

| | |on the number of transformations planned. |

| | |Only 0.1 ml cells is needed for each |

| | |transformation. |

|10. Leave these cells on ice for 1 – 2 hr until your ligation reaction is complete. | | |

|B. Partner #2 -Ligation of Your PCR Products to pCR2.1: You will now set up one ligation |( |Ligation reactions involving PCR products |

|reaction between the PCR products in your “FR” PCR tube and purified DNA of the vector | |must be carried out within 24 hrs of the |

|pCR2.1. | |completion of PCR, so the extra “A’s” added |

| | |to the PCR products by Taq will not be |

| | |cleaved off. |

|1. Put on a fresh pair of gloves and obtain another Isotherm and some ice. | | |

|2. Take your Isotherm to retrieve the strip of eight tubes containing your PCR products |( |Keep these tubes on ice at all times. |

|from the front table. | | |

|3. Use your “FR” PCR products to set up a ligation mixture containing the following |( |DNA ligase, which is purified from |

|components (set the other seven tubes of PCR products aside for now) in the order listed | |bacteriophage T4, joins DNA molecules |

|below: | |together by forming phosphodiester bonds |

| | |between them. |

| 5.25 (l sterile H2O |( |Clear tube, blue dot |

| 0.75 (l “FR” PCR products (with “A’s” at 3’ ends) | | |

| 1.00 (l 10X ligase buffer (purple tube with black dot) | | |

| 2.00 (l pCR2.1 DNA (with “T’s” at 3’ ends) |( |Green 0.65 ml tube |

| 9.00 (l total volume | | |

|4. Close the cap of the microtube, flick the tube vigorously to mix and spin it briefly | | |

|in the microfuge. | | |

|5. Add 1 (l T4 DNA ligase (purple tube) to this tube, flick it to mix and pulse spin the | | |

|tube again. | | |

|6. You will now let this reaction incubate for 1 - 2 hr on your bench at room |( |Be sure to record the time that this |

|temperature. Two hours is preferred. | |incubation begins. |

|C. Preparation of Selective Plates: Each lab pair should obtain three LB + Amp + IPTG + |( |Ampicillin and kanamycin are antibiotics used|

|X-Gal plates (one black stripe, one green stripe, one light blue stripe and one dark blue| |to select Amp or Kan resistant cells, X-Gal |

|stripe) from the front bench. | |is an artificial substrate for |

| | |(-galactosidase and IPTG |

| | |(isopropyl-(-thiogalactopyranoside) is an |

| | |inducer of the Lactose operon. |

|1. Label the bottoms of these plates with your initials and the date. |( |Use a black marker for labeling. |

|2. Label two plates with “Lig”, your initials and the date. | | |

|3. Label the third plate “no DNA”, your initials and the date. | | |

|D. Preparing Your PCR Products for Electrophoresis: | | |

|1. Add 2.0 (l loading dye (clear tube, purple dot) to each of your eight PCR tubes. |( |Do not discard this tube of loading dye. |

|2. Flick the tubes with your fingers to mix in the dye and spin these tubes in the |( |This pulse spin will bring the sample to the |

|microfuge for a few seconds using black adaptors. | |bottom of the tube. |

|E. Preparing your E-Gel for Electrophoresis: You and your partner will use an e-gel for |( |Ethidium bromide is a mild carcinogen, so you|

|electrophoresis because they are relatively easy to use and run rapidly. E-gels contain | |should always wear gloves when working with |

|ethidium bromide, which intercalates into DNA and fluoresces under ultraviolet | |e-gels. |

|illumination. Prepare your gel as described on the next page. Directions will be next to | | |

|your e-gel base and also reviewed by Dr. Malatesta or Dr. Sliski before you begin. | | |

|1. Use scissors to open the package containing the e-gel. | | |

|2. For red bases, plug the E-gel Power base into an electrical outlet using the adaptor |( |Older black e-gel bases may also be used. |

|plug. | |Consult the manual for proper use. |

|3. With the comb in place, insert the gel into the apparatus, inserting the right edge | | |

|first. | | |

|4. Press firmly at the top and bottom to seat the gel in the base. |( |When the gel is seated correctly, you will |

| | |hear a snap and a steady red light will |

| | |illuminate. |

|5. The Invitrogen logo should be located at the bottom of the base, close to the positive| | |

|pole, as shown below. | | |

[pic]

|6. It is necessary to pre-run the e-gel for 2 minutes prior to loading your samples. | | |

|a. For red bases, press and hold either button on the Power Base until the red light turns to|( |The flashing light indicates that the |

|a flashing green. | |2–minute pre-run has started. |

| | | |

|1) When the pre-run ends, the current will automatically shut off, the flashing green light | | |

|will change to a flashing red light, and the Power Base will beep. | | |

| | | |

|2) Wearing gloves, press and release either button on the Power Base to stop the beeping. | | |

| |( | |

| | | |

| | |The light will change from a flashing red |

| | |to a steady red. |

|b. For black bases, pre-run the gel with the comb in place for 1 – 2 min at 60 – 70 V. Then | | |

|turn off the power supply. | | |

| | | |

|8. Use both hands to remove the comb by gently lifting it and rolling it slowly toward you. |( |Do this for both red and black bases. |

|9. Obtain one tube of the 123 Base Pair Ladder (yellow tube) from the front bench. |( |The 123 Base Pair Ladder is a mixture of |

| | |fragments that differ in size by 123 base |

| | |pairs. |

|10. Load 20 (l of the 123 bp Ladder (yellow tube) into lane 1 of your e-gel. | | |

|11. Load 20 µl of each soil PCR product sample into one well in the order in which the |( |Be sure to record the order in which your |

|primers are listed on p. 34. | |samples are loaded. |

|12. Be careful not to introduce bubbles while loading, as they will cause bands to distort. |( |You can avoid introducing bubbles into your|

| | |sample by setting your Pipetman to 20 (l, |

| | |which is the exact volume you want to load.|

|13. Add 20 (l H20 (clear tube, blue dot) to any empty wells. | | |

|14. For red bases, press the 30 min button to begin the run. |( |Check that the dye is moving out of the |

| | |well. |

|15. For black bases, run your gel at 60 to 70 volts for approximately 30 to 40 minutes until |( |Do not run longer than 60 minutes because |

|the blue dye touches the black label at the bottom of the gel. Turn off the power supply to | |longer run times will damage the gel. |

|stop the run. | | |

|16. While your gel is running, obtain and prepare two tubes for your transformation as | | |

|directed in Section F below. | | |

|17. After electrophoresis ends, remove the gel cassette from the apparatus. | | |

|18. Place the cassette on top of the UV transilluminator and take two photographs of your |( |The camera should be set to 4.5 (f stop) |

|gel, one for each partner. | |and 2 (an exposure time of 1/2 sec). If a |

| | |lighter exposure is needed, set the camera |

| | |to 4.5 and 1 (exposure time of 1 sec). |

|F. Transformation of DH5( with Your Ligation Reaction: The next step in cloning is to | | |

|incubate your ligation reaction with the competent DH5( cells you prepared earlier. | | |

|1. Obtain two clear 1.7 ml microfuge tubes and three LB + Amp + X-Gal + IPTG plates (one |( |These tubes are for your transformation mixes.|

|black, one green, one light blue, and one dark blue stripe on side) from the bench at the | | |

|front of the lab. |( |Label two plates “Lig” and the third “No DNA”.|

|2. Use a black marker to label the top of one tube with your initials, 7/11 (the date) and | | |

|“Lig” for ligation mix. | | |

|3. Label the top of the second tube with “No DNA”, 7/11 and your initials. | | |

|4. Obtain fresh ice if necessary. | | |

|5. Put these two transformation tubes in ice. | | |

|6. Add 40 (l sterile TE buffer (clear tube, red dot) to the tube you labeled “Lig”. |( |Keep this tube of TE. |

|7. Add all 10 (l of your ligation reaction to this tube and flick it gently to mix. | | |

|8. Add 50 (l sterile TE buffer (clear tube, red dot) to the tube you labeled “No DNA”. |( |Do not add anything else to this tube. |

|9. Gently flick your tube of competent DH5( and add 100 (l of cells to each transformation |( |Flicking the tube will bring the cells off the|

|tube. | |bottom of the tube. Do not vortex these cells.|

|10. Flick each transformation tube gently with your fingers to mix. |( |Keep these tubes in ice at all times. |

|11. Let these transformation tubes sit on ice for 30 minutes. | | |

|12. After the 30 min on ice ends, put both tubes into the 42o C water bath for 90 seconds. |( |This heat shock stimulates the cells to take |

| | |up DNA. |

|13. Use your P1000 to add 1.0 ml sterile LB growth medium (15 ml orange-capped tube) to these|( |Use sterile technique to avoid contamination. |

|two tubes and flick gently to mix the contents. | | |

|14. Leave these tubes on your bench at rt for 45 min. |( |This incubation allows for expression of the |

| | |plasmid’s antibiotic resistance genes. |

|G. Plating Your Transformation Mixes to Selective Plates: When the 45 min incubation ends, | | |

|follow the instructions below to plate your cells to the LB + Amp + X-Gal + IPTG plates. | | |

|1. Transfer a 150 (l aliquot from your “Lig” tube onto each of the two LB + Amp/X-Gal/IPTG | | |

|plates labeled “Lig”. | | |

|2. Sterilize your spreader and spread the 150 (l liquid across the surface of each plate |( |Touch the spreader to a clear spot on the |

|until all the liquid has been absorbed. | |plate before using it to spread the cells. |

|3. Transfer 150 (l from your “No DNA” tube onto the plate labeled “No DNA”. | | |

|4. Flame your spreader and use it to spread until all the liquid is absorbed by this plate. | | |

|5. Tape your three plates together, write your initials on the tape, and incubate these | | |

|plates upside down overnight at 37o C . | | |

|6. Leave your two microfuge tubes (labeled “Lig” and “No DNA”) on your bench overnight until | | |

|you are sure your transformation is successful. | | |

III. REFERENCES

Achenbach, L.A., Carey, J. and M.T. Madigan. 2001. “Photosynthetic and Phylogenetic Primers for Detection of Anoxygenic Phototrophs in Natural Environments.” Appl. Environ. Microbiol. 67: 2922 – 2926.

Amann, R.I., Ludwig, W. and K.H. Schleifer. 1995. “Phylogenetic Identification and In Situ Detection of Individual Microbial Cells without Cultivation.” Microbiol Rev. 59: 143 – 169.

Baker, G.C., Smith, J.J., and D.A. Cowan. 2003. “Review and Re-Analysis of Domain-Specific 16S Primers.” J. Microbiol. Meth. 55: 541 - 555.

Biological Procedures Online. bpo/arts/1/6/m6.htm .

Cole, J.R., Chai, B., Farris, R. J., Wang, Q., Kulam, S. A., McGarrell, D. M., Garrity, G. M. and J. M. Tiedje. 2005. “The Ribosomal Database Project (RDP-II): Sequences and Tools for High-Throughput rRNA Analysis.” Nucleic Acids Res. 33: D294 – D296.

Epicentre Biotechnologies ().

Handelsman, J. 2004. “Metagenomics: Application of Genomics to Uncultured Microorganisms.” Microbiol. Mol. Biol. Rev. 68: 669 – 685.

Janssen. P.H., Yates, P.S., Grinton, B.E., Taylor, P.M. and M. Sait. 2002. “Improved Culturability of Soil Bacteria and Isolation in Pure Culture of Novel Members of the Divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia.” Appl. Environ. Microbiol. 68: 2391 – 2396.

Kuske, C.R., Barns, S.M. and J.D. Busch. 1997. “Diverse Uncultivated Bacterial Groups from Soils of the Arid Southwestern United States That Are Present in Many Geographic Regions.” Appl. Environ. Microbiol. 63: 3614 - 3621.

Milling, A., Smalla, K., Maidl, F.X., Schloter, M. and J.C. Munch. 2004. “Effects of Transgenic Potatoes with an Altered Starch Composition on the Diversity of Soil and Rhizosphere Bacteria and Fungi. Plant & Soil 266: 23 – 39.

Rappe, M. S. and S. J. Giovannoni. 2003. “The Uncultured Microbial Majority.” Annu. Rev. Microbiol. 57: 369 - 394.

Rondon, M.R., August, P.R., Betterman, A.D., Brady, S.F., Grossman, T.H., Liles, M.R., Loiacono, K.A., Lynch, B.A., MacNeil, I.A., Minor, C., Tiong, C.L., Gilman, M., Osburne, M.S., Clardy, J., Handelsman, J., and R.M. Goodman. 2000. “Cloning the Soil Metagenome: A Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganisms.” Appl. Environ. Microbiol. 66: 2541 – 2547.

Schloss, P. D. and J. Handelsman. 2004. “Status of the Microbial Census.” Microbiol Mol. Biol. Rev. 68(4): 686 - 691.

White, T.J., Bruns, T., Lee, S. and J. Taylor. 1990. “Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics.” In PCR Protocols, A Guide to Methods and Applications, pp. 315 – 322. Edited by M. Innis, D.H. Gelfand, T.J. Sninsky and T.J. White. Academic Press. San Diego, California.

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.

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