Identification of Unknown Microorganisms by 16S rDNA Sequencing

I. Abstract:

The identification and study of various microorganisms is an essential procedure for living a healthy and well-balanced life. From medical diagnostics to the food industry, identifying microorganisms is extremely important when identifying pathogens as a cause of disease or identifying microbial contaminants in food. For this experiment, the identification of microorganisms was done using 16s rDNA. For this process five steps were initiated: DNA isolation, PCR amplification of the 16s rDNA gene, gel electrophoresis as well as purification of the 16s rDNA, followed by 16s rDNA gene sequencing, and lastly a BLAST analysis was conducted to determine the identification of the unknown. In accordance to the BLAST databases, our unknown # was most comparable to both Pseudomonas aeruginosa and Pseudomonas resinovoranswith abit score of 604 and contained an e-value of 7e-173 for both strains.

II. Introduction:

Prokaryotes and eukaryotes ribosomes contain a complex structure in which it is able to synthesize proteins with. In prokaryotes the ribosomes are made up of 50S large subunit as well as 30S small subunit, added up this makes up the 70S ribosome. In addition, the 50S large subunit contains both the 5S and 23S ribosomal RNAs (rRNA) and the 30S small subunit is composed of 21 polynucleotide chains and16S rRNA. The16S rRNA is encoded by the16S ribosomal DNA (rDNA) ². The 16S rDNA is highly conserved (least variable) between different species of bacteria and archaea, however it also contains hyper variable regions in which are accumulated through evolution². These random sequence changes vary among species, which enables them to identify and determine different bacterial species from one another¹. The 16S rDNA sequence has been extensively used by research studies to determine taxonomy among species, phylogenetic relationships and estimate species divergence, and in this lab specifically for identifying unknown organisms².

In addition, a technique performed to determine the bases that make up a genetic sequence of a specific sample is DNA sequencing. However, in order for DNA to be sequenced, through Polymerase Chain Reaction (PCR) enough copies of the observed DNA need to be produced¹. Through the use of PCR as well as 16S rDNA, the identification of unknown organism can be determined. In DNA sequencing, 16S rDNA is recommended for the reason that the rDNA can be located in all cells, the genes are highly conserved, and abolishes the requirement of microbial cells culturing².

Furthermore, technique of PCR is widely used because it is a fast and inexpensive way to amplify small segments of DNA. ¹In this process sufficient amounts of DNA are required for genetic as well as molecular analyses. Using the polymerase chain reaction techniques, one can easily and rapidly amplify certain segments of DNA and obtain millions of copies of the DNA segment³. Through the amplification of DNA many uses can be made for a variety of different testing such as sequencing, fingerprinting, diagnosing genetic disorders and of course the identification of organisms. 

Polymerase Chain Reaction PCR is a three-step process; the first step is denaturation, annealing, and finally extension¹. In the denaturation step the breaking of hydrogen bonds between nitrogenous bases must be made. Here the DNA sample is heated to approximately 95°C per minute which separates the DNA double strands into two single strand pieces of DNA¹. Next, the temperature is decreased to approximately 55°C per minute, enabling DNA primers to bind to their complementary DNA sequences². The primers consist of short, 20 base pairs long, DNA single stranded pieces that are complementary to the ends of the target DNA sequence³.Universal primers used to detect the 16S rDNA gene of the majority of bacterial speciesare DG74 and RW01. The DG74 and RW01 primer amplifies fragments of 370 bp PCR products². After the binding of the primer to the target DNA, Taq polymerase synthesizes into two new strands of DNA between 72°C for 1 minute since around 1000 base pairs bind together during 1 minute of amplification and extensionbecause of the enzymes ability to isolate from bacterial thermophiles that can withstand high temperature. A semiconservative replication of the original DNA strands is made as a result which can act as templates for the next round of DNA amplification. Then the steps are performed again about 30 times and once DNA amplification is completed, finally the temperature will be held at 72°C for approximately 10 more minutes to be able to amplify any remaining unfinished PCR products². The entire PCR process is performed in a thermocycler that is programmed to alter the temperature of the reaction every few minutes as indicated to allow DNA denaturing and synthesis. Once the final products of a PCR reaction are obtained they can finally be analyzed through gel electrophoresis and DNA sequencing. Through the aid of BLAST which stands for the basic local alignment search tool we are able to compare primary biological sequence information, like the amino-acid sequences of proteins or the nucleotides of DNA/RNA sequences. BLAST first detects local alignments in comparison to each sequence and determines which one works the best, starting off with a small set of three letters, which they call the “query word.” The query word represents the three amino acids or nucleotides, in a certain order3.

III. Materials and Methods:

In this experiment, an unknown microorganism culture which was left overnight and was resuspended in which 1.5 mL of the culture was transferred into a sterile microcentrifuge tube. Using a micro centrifuge the culture was then left to centrifuge at 13,000 rpm for 5 minutes and the supernatant was then removed without disturbing the cell pellet¹. Next, the cell pellet was resuspended with 180 µl of Lysis buffer and incubated at the specific temperature of 37 °C for approximately 30 minutes. Once the incubation was complete, 200 µl of buffer AL as well as 20 µl of proteinase K were added into the microcentrifuge tube and then vortexed for about 10 seconds.Then, the sample was incubated at 56 °C for another half hour (30min) and centrifuged for 5 seconds at a max speed. Next, ethanol was added to the sample and once again vortexed for 15 seconds¹. The sample was transferred into a QIAamp spin column and centrifuged at 8,000 rpm for 60 seconds¹. This was to allow the genomic DNA to be able to stick to the column and for the supernatant to flow through and be discarded and as well as reassemble the column to the same collection tube ².Then, 500 µl of AW1 buffer was added and the sample was centrifuged at 14,000 rpm for 3 minutes which was used to rinse the DNA and remove any soluble impurities². The flow through was discarded and the collection tube was spun for one minute to allow the column². Next, the spin column was placed in a 1.5 ml microcentrifuge tube and 100 µl of AE buffer was added into the column¹. After a minute, the sample was centrifuged at 8,000 rpm for 1 minute causing the purified DNA to end up in the collection tube². Finally, a nano-drop machine was used to determine the concentration of genomic DNA and the purified DNA was marked and stored in the refrigerator at -20 °C until the following portion of the lab experiment could be carried out².

The next part of the lab we received back our pure DNA that was isolated and the concentration of the genomic DNA was indicated on the tube. If the DNA concentration was not equal to 100 ng/µl, the volume of isolated genomic DNA needed to get 100 ng once calculated². The PCR reaction was then set up by adding reagents to the unknown sample in the following order: dH₂O(25 µl), primers (1.25 µl RW01 and 1.25 µl dg74), DNA(5 µl), and Master Mix (12.5 µl)². The volumes of reagents differed to those in the lab manual and were reduced by half due to us not having enough of the reagents for the entire class. As soon as the set up was complete, the instructor initiated PCR and the product was used in the following weeks lab.

The following lab consisted of us receiving the PCR product and 30 µl of it being transferred into a sterile 1.5-µl microcentrifuge tube along with 30 µl of loading buffer¹. The contents of the tube were mixed prior to the 30 µl being transferred into the designated well into the agarose gel². The process of loading the wells were as followed: 10 µl of 100 bp ladder which serves as a DNA marker, 8 µl of the positive DNA control, 10 µl of the negative DNA control, 13.75 µl of Sample #1, as well as the same amount of µl as the other unknown samples from the remaining groups. The gel was then ran at 100 volts for 30 minutes giving the orange dye to migrate approximately two-thirds down the gel¹. After electrophoresis had met completion, the gel was observed under the aid of the UV transilluminator². Then, using a razor blade the 370 bp PCR fragment was cut from the agarose gel and placed into a 1.5 mL microcentrifuge tube, which we had weighed prior to use².

Using the QIAquick gel elution system the PCR fragment was then purified. Next, we weighed the microcentrifuge tube containing the sample and the weight was subtracted from the initial weight of the empty tube. This provided us with the weight of the sample¹. Next, 3 parts of QG buffer was added for every 1 part (0.1 g or 100 µl) of fragment¹. The micro-centrifuge was then placed in an incubator for 10 minutes at 50 °C for 10 this way the fragment would fully dissolved in the QG buffer². Next 1 gel volume of isopropanol was added and mixed into the sample². The sample was then applied from the microcentrifuge tube to the QIAquick column and centrifuged for 1 minute¹. About 0.75 mL of PE buffer was added to the sample, and the flow through was discarded. The sample was centrifuged for 1 minute to wash the column and the flow through was discarded again¹. By centrifuging the column it had dried. Next, the column was placed in a 1.5 mL microcentrifuge tube and 30 µl of EB buffer was added to the center of the column to elute the DNA². The sample was centrifuged for 1 minute so that the eluted DNA could be collected from the bottom of the tube². Lastly, the sample was given to the instructor and ready for sequencing².

Lastly, for the final part of the experiment, BLAST (Basic Local Alignment Search Tool) was used to determine the identity of the given unknown organisms¹. Once on BLAST, the Nucleotide Blast option was chose to be granted access to databases that contained the 16S rRNA sequences of Bacteria as well as Archaea. The sequencing data received for the given unknown organism was inputted and searched for on BLAST and the once detected the local alignments between sequences that work the best were the primary hit that identified the unknown organism ¹.

IV. Results:

Table 1: Number of Unknown Sample and Purity of Genomic DNA

(A260 nm =1 indicates that there are 50 µg/ml of double stranded DNA in the solution)

Table 1: The table located above displays the number of the unknown that was given, which was unknown sample number one. It also shows the concentration of the DNA that was isolated from the unknown, which was found using a nanodrop machine, which measured the absorbance of the sample at 260/280 nm². The concentration of DNA in the sample is correlated to the absorbance of the sample at 260/280 nm².

Figure 1.

Figure 1: The picture above shows the electrophoresis result of the PCR product. To the farthest left is the 100 bp ladder followed by its negative and positive control on the 2^nd and third lane. The 4th lane indicates another groups unknown and lane 7 also is another 100 bp ladder with a positive and negative control on lanes 8 and 9 followed by another groups unknown on lane 10. The 5^th lane however indicates our gel from the unknown sample #1 at band 370 bp which is shown by the neon orange light, located a third down the gel.

Blast Analysis

Figure 2: This is the Blast description box that indicates BLAST’s top hits along with the bit score and the e value. The bit score for unknown sample #1 is 604 and has an E-value for Pseudomonas aeruginosa and Pseudomonas resinovorans, indicating they are both the top hits.

Figure 3.

Figure 3:In the image above it displaysthe two top DNA sequence alignment between the query and the subject. Two are shown for the reason that two microorganisms belonging to the bacteria/ archaea group have a extremely similar sequence in comparison to the unknown sample #1.

The results we obtained for the unknown sample # 1 was the concentration which was 5.4 ng/µl by using the purity of 2.45 at the absorbance of A260/280 nm which was given by our professor, and manipulating the equation.  From the gel electrophoresis we were able to match the ladder band at 370 bp on the far left to that of our unknown sample #1 which is displayed by the bright orange band on lane 5. For the BLAST analysis we were directed to the link located in our lab manual: https://blast.ncbi.nlm.nih.gov/Blast.cgi 

There we were able to input our DNA sequence given to our professor through the results of the gel. The results that we obtained were Pseudomonas aeruginosa and Pseudomonas resinovorans, in which were both the top hitswith abit score of 604 and contained an e-value of 7e-173 for both. Figure 3. Shows the RNA sequence of both Pseudomonas aeruginosa DSM 50071 16SRibosomal RNA and Pseudomonas resinovorans ATCC 14235 16S Ribosomal RNA,along with their lengths and the alignment the two contain between the sample #1.

V. Conclusion and Discussion:

In conclusion the BLAST E-value is the number of expected hits , indicating that of a similar quality (score) that is found by chance. An E-value of 10 means that up to 10 hits can be expected to be found, given the same size of a random database3 .The way Blast works is that the results are sorted by E-value by default which means that the best hit is in first line. Which in our case would be Pseudomonas aeruginosa but on the second line was Pseudomonas resinovorans with the same exactbit score of 604 and contained an e-value of 7e-173 for both strains. We found this by the sequence given to us by our professor and then inputted the sequence on the given site. The highest bit score indicated that our unknown #1 and Pseudomonas aeruginosa and Pseudomonas resinovorans are the most similar. In addition,the high e-value indicates that the unknown sample #1 didnt indicate any evolutionary relationship with the two. Where as the 99.40% identity suggests that the nucleotides percentage of the unknown and the two microorganisms are the the best related matches. Furthermore, in regards to the data resulted from this lab experiment, the extracted DNA concentration for our sample #1 was 5.4 ng/µl with a purity of 2.45 for absorbance A260/A280. However, indicated by the lab manual the purity of A60/280  ratio of 1.7-2.0 indicates pure DNA suitable for this molecular biology experiment, although we obtained a higher purity. Having said that there must have been presence of RNA as contamination.

VI. References:

1. CSU Faculty. Micr 3100: General Microbiology Laboratory Exercises. N.p.: Print. Fall 2018.
2. Polymerase chain reaction (PCR). Khan Academy. [accessed 2018 Nov 16].  https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/a/polymerase-chain-reaction-pcr 
3. Raengpradub, Sarita. “Microbial Identification: Tracking the Great Unknown with Innovative and Advanced Technologies – Sarita Raengpradub, 2009.” SAGE Journals, journals.sagepub.com/doi/full/10.1016/j.jala.2008.12.011.