1. Isaac Cao, Theoneste Hakorimana and Kurtis Nokes
PARE Project
Isaac Cao, Theoneste Hakorimana and Kurtis Nokes
2018 Fall Semester
Kergerye

2. Introduction
The PARE project is a citizen-science research project that records and monitor antibiotic resistances organism. In other words, we are trying to find organisms that are resistance to antibiotic in our soil. From doing this research project, we are able to provide an in-depth contribution to not only for scientific community, but as well as in the health community.

3. Soil Sample Collection Data
The soil we collected was at a park that have frequent visitors that comes and goes to relax and have fun. Since, this area is visited so much, there are bound to have people who play in the dirt and bring unwanted guest at home or anywhere else.
Fassio Farm Park, 3720 So W, WVC, UT 84120
Latitude: & Longitude:
Town park/Playground/Dog park
Collection Date: 1130 with Cloudy weather condition

4. PARE Data Results Colony collected Colony Collected
5 day period before plated
Culture medium: lMAC
Incubation temp: 28 C
Hours of incubated: 72 hours
Used Tet3 (10^3) for set 1 and 2 for Agarose Gel Electrophoresis

5. PARE Data result continue
Percent tetracycline resistant cells
CFUs Per gram soil
Plate set 1
Plate Set 2
Plate 1
Plate 2
# Colonies
216 68
Volume of Cell plated
0.2 mL
Dilution Factor
10^4
10^5
Total CFU/gram soil
1.08x10^7
3.4x10^7
Tetracycline concentration
3 ug/ml
30ug/ml
30 ug/ml
# Colonies
210 58
227 38
Volume
0.2 mL
Dilution Factor
10^3 10
Total Tet CFU/gram soil
1.05x10^6
2.9X10^3
1.135x10^6
1.9x10^3
Percent of Tet CFU
9.72%
0.027%
3.36%
0.0056%
Table 1
Table 2

6. Agarose Gel Electrophoresis Results
Results are on the Second row
Well 2 (17433) for set 1
~750 BP
Well 3 (17434) for set 2
<50 BP
Both got analyzed

7. DNA Sequencing Results
Set 1: 17433
1000 in length, ~750 Base Pairs
**NNNNNTCNANNGNGGTACCGTCCTCCCGAAGGTTAGACTAAGCTACTTCTGGTGCAA CCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGAC ATTCTGATTCGCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATC CGGACTACGATCGGTTTTATGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTTTGTACC GACCATTGTAGCACGTGTGTAGCCCAGGCCGTAAGGGCCATGATGACTTGACGTCATCC CCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAGAGTGCCCACCATAACGTGCTGGTA ACTAAGGACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCT GACGACAGCCATGCAGCACCTGTCTCAATGTTCCCGAAGGCACCAATCCATCTCTGGAAA GTTCATTGGATGTCAAGGCCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGC TCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCC CAGGCGGTCAACTTAATGCGTTAGCTGCGCCACTAAGAGCTCAAGGCTCCCAACGGCTA GTTGACATCGTTTACGGCGTGGACTACCANGGTATCTAATCCTGTTTGCTCCCCACGCTTT CGCACCTCANTGTCAGTATCAGTCCNGTGGTCGCCTTCGCCACTGGTGTTCCTTCCTATAT CTACGCATTTCACCGCTACACANGAAATTCCACCACCCTCTACCANACTCTAGCTTGCCAG TTTTGGATGCANTTCCCAGGTTGAGCCCCGGGATTTNNCATCCCACTTAANNAAANNNC CTACGCGCGCTNACGCCAGTAATTCCNANNAANGCNTGCACCCCNCTGNANTACCGCN NNGCNGGCNNNNNNNNNNNNNNGCTNATTCTGTNGNANNTNNNNN**
Set 2: 17434
58 in length, Less than 50 Base Pairs
Not enough base pair to run the BLAST program. Most likely cause of the small sequences can be that we did not put in enough of our colony in the PCR solution for Set 2.
**NNNNNNANNTNNNNNNNNNNNNNNNNNTNATCTGAGCCATCGNTCTAA CNNNNNNNCT**

8. BLAST Results and Identification
Uncultured Bacterium gene for 16S rRNA partial sequence, clone: HSM050P-B-4
Query Cover: 94%, E value: 0, Ident: 97%
PCR-derived sequence from peat soil (Turf); accumulation of partially decayed vegetation or organic matter.
Masaru Akiyama Horonobe Research Institute for the Subsurface Environment (H-RISE), Northern Advancement Center for Science & Technology; Sakae-machi 5-3, Horonobe, Hokkaido , Japan

9. Phylogenetic Tree
Specimen that are closely match for BLAST results

10. Pseudomonas sp. 55A1
Pseudomonas sp. 55A1 16S ribosomal RNA gene, partial sequence
-Second Match on BLAST w/ a name of an organism
Query Cover: 94%, E value: 0, Ident: 97%
Proteobacteria; Gammaproteobacteria; Pseudomonadales; Pseudomonadaceae; Pseudomonas.
Becker,M.H., Walke,J.B., Murrill,L., Woodhams,D.C., Reinert,L.K., Rollins-Smith,L.A., Burzynski,E.A., Umile,T.P., Minbiole,K.P. and Belden,L.K.

11. Summary
From obtaining soil to analyzing it genetic code we were able to learn so much regarding our project. We were able to decoded one of our colony, and learn that this organism is resistant to a low dose of the tetracycline antibiotic. In addition, as we increase the dose of the tetracycline, we see a decrease trend in colonies being formed. So, if we have kept going with increasing the dose on our plate then we would have seen fewer and fewer growths in the plates. Even though we were able to decode one of our colony we were able to learn so much about it on the BLAST search. From the people who discover the organism to the family tree of that organism. From all this we were able to learn that even a local park can contain antibiotic resistant organism.
So, let your biology department, and even your local high school to get involved in the PARE project. If we increase our knowledge of antibiotic resistant organisms we may benefit not only as a community but as a whole nation. As knowledge is power and with that power we can spread and teach others on how to be safe against these organisms. In addition, provide more resources so that future scientist we could try this experiment and hopefully use different antibiotics than the tetracycline to see if any other organisms are resistant. From this we can improve the PARE database and the near future could be uses for future experiments with antibiotic resistance organisms.
In conclusion, this research project was able to provide an in-depth contribution to not only for scientific community, but as well as in the health community. As we hope that this information will teach you how to be safe against these organisms.

12. Interesting Website regarding the Organism
-Antibiotic resistance in Pseudomonas Research-
-Sign and Symptoms of Pseudomonas Infections-
-Antibiotic resistance Research on Gammaproteobacteria
-Increase trend of Antibiotic Resistant Organism