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Obtaining Bacillus Luciferensis

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ABSTRACT

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Two people decided to find out what types of bacteria is growing in campus. Soil was the choice of sample gathering. The bacteria was obtained and cultured to give rise to colonies which was then taken to determine its morphology as well as to get a pure stock culture. Some of the stock culture was then centrifuged to obtain a pellet of bacteria cells and then its DNA was obtained by lysing the cells. After DNA purification, it was sent for PCR. The PCR was then sent for sequencing. Then inferring from the sequence, the bacteria was identified as Bacillus luciferensis.

INTRODUCTION

At NYP campus, in fact anywhere in the world, there are microorganisms everywhere. From phone surfaces, to the sewers and even the inside of bleach containers, bacteria is present. The study was carried out to determine what kind of bacteria is present on campus and to find out more on the acquired bacteria. More specifically, this study was conducted to find out what bacteria is present in the wildlife in NYP campus, what types of bacteria are growing in there and if there are any interesting bacteria that has not been well studied on. To do so, a soil sample was taken from NYP campus and a colony was obtained.Its DNA was extracted and sent for sequencing to determine the bacteria. After sequencing, the nucleotide sequence was then compared with the database of bacteria in NCBI {2} and discovered that the bacteria obtained was bacillus luciferensis. After finding information about the obtained bacteria, it possesses the following properties; it is a gram variable rod-shaped bacteria measuring about 3-6μm by 0.4-0.8μm. Colony appeared an irregularly shaped creamy grey colony that was translucent. colony size measures about 1-5mm in diameter and is also found to be a facultative anaerobe. As it is part of the bacillus genera, it is able to form endospores. It was found in geothermal soil from an active fumarole on lucifer hill, candlemans island located in antarctiica found in 1998 {1}.

METHODS

Collection of sample and culturing sample

Soil sample was collected at the plants outside block S in NYP campus. Next, a 7X serial dilution was performed on the soil sample and the 1x102X and 1x106X diluted sample was cultured on an agar plate by spread plate method. This plate was then incubated at 37 °C. After incubation, multiple colonies were formed on the 1x102X plate as seen in Figure 1. while no colonies were formed on the 1×106 X plate

Gram staining

A small part of a raised, creamy-gray colony was obtained from the culture. This was followed by mixing with a small drop of water on a microscope slide. The slide was heat-fixed by running through a bunsen burner 3 times and left to air dry. Then, it was viewed under a microscope up to 1x103X magnification. The bacteria slides could be seen in Figure 2.

Culturing a stock solution of bacteria

The same colony form the gram stained colony and cultured in nutrient broth by taking a micropipette tip and scraping on the selected colony. The bacteria filled micropipette tip is then placed in the vial of nutrient broth and left to incubate.

Obtain a pellet of bacteria via centrifugation

After incubation, the nutrient broth was turbid, indicating there was bacterial growth. 800μl of the stock solution of bacteria was put in an eppendorf tube by extracting it from the stock solution. The sample was centrifuged and the supernatant was discarded, leaving the pellet at the bottom. If the pellet was too small, more stock solution could be added, centrifuged to get a larger pellet.

DNA extraction from cell lysate

DNA was extracted from cell lysate as per PureLink™ genomic kit protocol. The pellet was then resuspended in 180μl Purelink Genomics Lysis buffer. 20μl of proteinase K that was provided along with the lysis kit was added to lyse the cells. Mix the tube well by brief vortexing. Then, the eppendorf tube was incubated at 55℃ with brief vortexing for every 5 minutes. This is done for 30 minutes.Then, 20μl of RNAase A while briefly vortexing it and incubate for 2 mins. Then, a master buffer was made by adding 200μl of purelink lysis buffer and 200μl of 96-100% ethanol. 400μl of the master buffer is added to the lysate and mixed well by vortexing.

DNA purification using spin columns

DNA was purified as per PureLink™ genomic kit protocol instructions, about 640μl of lysate was added to the spin column, along with Purelink Genomics lysis buffer and ethanol. The column was then centrifuged at 1×104 x g for 1 min at RT. the collection tube was then discarded and the spin column was placed in a new collection tube that was supplied with the kit. Then, 500μl of wash buffer 1 was added to the column, followed by ethanol. The column was then centrifuged at 1×104 x g for 1 minute and the collection tube was discarded. The spin column was then placed in a new collection tube and 500μl of wash buffer 2 was added to the spin column, along with ethanol. The spin column was centrifuged at maximum speed for 3 minutes at RT. the collection tube was then discarded. Then, the spin column was placed in a sterile 1.5-ml microcentrifuge tube and 30μl of purelink genomic elution buffer was added to the spin column. The spin column was then left to incubate at RT for 1 minute before centrifugation at maximum speed for 1 minute. The microcentrifuge tube now contains purified genomic DNA. A gel electrophoresis was performed to test if DNA extraction was successful. To test for the presence of DNA in the sample, 3μl of the DNA containing elution was added to 2μl of distilled water and 1μl of 6X loading dye The results can be seen on Figure 3.

PCR purification and sequencing

As per PureLink™ quick gel extraction kit protocol instructions, a segment of the gel that contained the DNA of interest was excised weighed using a scale sensitive to 0.001g, put into an eppendorf tube, followed by the addition of gel stabilization buffer (L3) into the tube in a 3:1 ratio whereby 71.7 mg of gel was used, therefore 215.1 ml of buffer L3 was added. The tube was then placed in a 50 C heat block for 10 minutes while inverting the tube every 3 minutes to ensure gel dissolution. Upon the dissolution of the gel piece, the tube is then incubated in the heat block for an additional 5 minutes. The dissolved gel containing genomic DNA in the tube was then transferred into a spin column. The spin column was then centrifuged at >1.2×104 x g for 1 minute and the collection tube containing the flow-through is discarded. 500μl of wash buffer (W1) containing ethanol was then added to the column. The spin column was then placed in a new collection tube and centrifuged at >1.2×104 x g for 1 minute to remove the buffer. The collection tube was discarded and replaced with a new one, then the spin column was centrifuged at maximum speed for 1-2 minutes to make sure there is no more ethanol in the spin column. The flow-through in the collection tube was then discarded and replaced with a new collection tube. 50μl of elution buffer (E5) was added to the center of the spin column and incubated for 1 minute at RT to allow the elution buffer dissolve the DNA that is within the matrix filter. The tube was then centrifuged at >1.2×104 x g for 1 minute and the elution containing the DNA was collected and sent for sequencing

RESULTS

OBTAINING COLONIES GRAM STAINING

DNA PURIFICATION PCR

DISCUSSION

After culturing cells, there are multiple colonies in the 1x102X culture plate. There are not only bacterial colonies but also fungal growth in the plate. A colony was carefully chosen and picked up to be cultured in nutrient broth for further use.

From the results of the gram staining, the colour of the cells indicate gram-negative cells. However, from reports of Bacillus luciferensis, it is typically a gram-positive bacteria. It is deduced that B. luciferensis is a gram-variable microorganism that becomes gram-negative after 24 hours of culture at 30°C and above. Since it was cultured for more than 24 hours, gram-negative morphology can be explained.

After DNA purification, the sample was faintly seen on the gel (Fig 3.). Presence of genetic material is typically indicated with a bright band. However, in this gel it is faint. This shows that there is little genetic material in the sample after purification. This can be explained by insufficient genetic material during gel electrophoresis. During the mixing of loading dye and genetic material, the mixture was left out too long and was stuck to the wall of the pipette tip leaving insufficient mixture to go into the gel, hence had to be re-done. The band might not be as bright due to too much loading dye in comparison to DNA. The DNA purification could have been done improperly, hence showing only a faint band on the gel. Genetic material taken for gel electrophoresis might not be the accurate amount, hence showing a faint band on the gel.

When making the master mix for PCR reaction, 9 sets were run for the bench for 7 samples and 1 set was for the negative control to be run. One extra set was also run so as to eliminate the carry-over effect whereby liquid is lost when stuck to the sides of the eppendorf tube. The negative control is inclusive of everything in the master mix except for DNA, which was replaced with distilled water. This was run on the gel to ensure that there was no contamination for any components in the master mix. Should the band light up for the negative control, the master mix is contaminated. PCR was carried out to amplify the genetic material to send for sequencing. 25µl of PCR product was mixed with 5µl of loading dye, and 28µl of the mixture was used for gel electrophoresis in order to prevent a carry-over effect from occuring as much as possible. The rest of the DNA that was not used in the PCR reaction was stored at 4°C. It can be stored for safety purposes, in the case that the PCR reaction does not work as well as intended.

One of two samples that was initially cultured, purified and has undergone PCR has not shown a band in the second gel electrophoresis that was carried out after PCR (Fig 4.). However, there was a band in the gel electrophoresis after DNA purification. (Fig 3.) This is an indication of the presence of DNA. However, there is absence of genetic material after PCR is performed. This can be explained by damaged, poor quality template, or impure DNA. There could also have been a contamination while doing the first gel, which shows the presence of DNA in the gel. However, this may not have been the DNA of the bacteria that was initially cultured. Since the genetic material was found to be an rRNA, it could have been degraded after purification and when being stored. This could have resulted in degraded rRNA running for PCR. Hence possibly explaining why there was no band in the second gel electrophoresis after PCR was performed.

There were two gels run, once after DNA purification and another after having run PCR. A gel electrophoresis was run after DNA purification to determine the presence of DNA before proceeding with PCR. The second gel electrophoresis was carried out to confirm the presence of DNA in the PCR product before proceeding with further DNA purification after the excision of the gel containing the band.

After sending the purified product for sequencing, a computer program, FinchTV, was used to view the chromatography of the sequence (See Appendix A). On FinchTV, the first few nucleotides and the last few nucleotides were removed due to weak peaks. This was done to ensure increased accuracy of the nucleotide sequence, as the first few nucleotides were not as accurate as those in subsequent nucleotides. The first 25 and last 2 nucleotides were removed, leaving a 229 nucleotide long sequence (See Appendix B) to be cross-matched on NCBI Blast to discover that the bacteria obtained was Bacillus luciferensis.

CONCLUSION

The purpose of this lab was to find out what kind of bacteria is present in the NYP campus, more specifically in the soil outside block S. The soil sample was serially diluted, cultured, and then extracted, purified and amplified. It was sequenced and the bacteria was found to be Bacillus luciferensis. Further research on Bacillus luciferensis was done and found that it is a facultative anaerobe and has the ability to form endospores, as all bacteria from the bacillus family should. It is a mesophile that grows under an optimal Ph of 7.0 and the Ph range at which it can survive is 5.5-6.0, has a optimal temperature for growth at 30????C with a range from 15-45????C and also has a G-C content of 33% {1}. It

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