• The following naming scheme was used throughout the experiments unless specified otherwise.
• Tubes are labeled with the format “Db#MP”
• Plates are labeled with format “Db#T-MP”
  • D represents the origin of the bacteria which was soil
  • b represents the original bacteria growth used
  • # represents the number of weeks the bacteria had previously grown in a tube with the initial bacterial growth starting at zero
  • M represents the media the bacteria was grown in while in a tube
  • P represents the pesticide used during all steps of growth
  • T represents the technique used to apply the bacteria to the plates
• Grid plate tubes were labeled AP EG 1-10 with the following system showing what quadrant plate square was used.
  • 1-5 = px
  • 6-10 = mp
  • 1,2,6,7 = Db3Q
  • 3,4,8,9 = Db4Q
  • 5,10 = Db5Q
  • Even = c
  • Odd = s
  • Exception: 5 being for Db5Qcpx

02/04/2021

• Approximately 10 g of soil sample was collected 1-foot underground at Latitude: 43.206349 Longitude: -77.770677

• This was collected at night and kept in an opaque box in order to limit the light exposure.
• The soils initially were stored at room temperature in a dark drawer for 48 hours before being relocated to a dark fridge.

02/05/2021

• 1 g of soil was put in a tube with 10 µL of all the media and flipped 10 times before being incubated 35°C for 48 hours with no light.
• This was labeled as “Dirt 0”

02/07/2021

• 7 mL of the “Dirt 0” supernatant was put in a tube with 3 mL of LB minimal media. The tube was then vortexed then incubated in the dark at 35° in a shaking incubator for 48 hours at 200 RPM.
• This was labeled “D1”
• It was later determined that 7 mL of “Dirt 0” was too much bacteria to LB ratio.
• The image below is of the resulting tube.

02/09/2021

• Steps from 02/07/2021 were repeated with 170 µL of “Dirt 0” and 10 mL of LB instead.
• This tube was labeled “Db1”
• The image below is of the resulting tube.

02/19/2021

• For the next step, four tubes were created with the goal of testing our bacteria medium from “Db1”(which was mislabeled as “D1b”) against the two pesticides paraoxon(PX) and methyl parathion(MP) on both CSM media and SSM media.
• First 5 mL of the respective media was put into a tube
• Next 170 µL of “Db1” was put into each of the four tubes.
• Then 5 µL of respective pesticides were added last and only under a hood.
• Due to the shaking incubator not being adjustable to the correct temperature, the tubes were put in the fridge wrapped in aluminum foil until 2/25/2021.
• They were then put in the incubator at 30°C at 200 rpm in the dark until 3/4/2021.
• The images below are of the resulting tubes.

03/04/2021

• During the incubation of “Db1” discovered that two errors had occurred on 02/19/2021. The first error was that two tubes were mislabeled with either the wrong pesticide or the wrong media. Furthermore, pesticide concentrates were used instead of the intended pesticide dilution. For both these reasons this portion of the experiment was redone with the proper corrections.
• All of the following components were added to the tube under a hood wrapped in aluminum foil.

• 5 mL of the respective media
• 170 µL of “Db1” into all four tubes.
• 5 µL of respective diluted pesticide
• All tubes were then put in the incubator at 30°C at 200 rpm in the dark until 3/11/2021.
• The images below are of the resulting tubes.

3/11

• All the same steps as on 03/04/2021 with the difference being
  • The tubes incubated until 03/18/2021
  • Each tube instead sampled from the previous step with the same media and pesticides.
    • For example “Db3cm” sampled its bacteria from “Db2cm”.
• The images below are of the resulting tubes.

3/18

• All the same steps as on 03/04/2021 with the difference being
  • The tubes incubated until 03/25/2021
  • Each tube instead sampled from the previous step with the same media and pesticides.
    • For example “Db4cm” sampled its bacteria from “Db3cm”.
• The images below are of the resulting tubes.

3/25

• All the same steps as on 03/04/2021 with the difference being
  • The tubes incubated until 04/1/2021, in which the tubes were temporarily put in the fridge until later experimentation.
  • Each tube instead sampled from the previous step with the same media and pesticides.
    • For example, “Db5cm” sampled its bacteria from “Db4cm”.
• The images below are of the resulting tubes.

4/2

• 100 µL of pesticide was spread onto minimal media plates and dried under a hood for two hours
• 100 µL of the respective liquid cultures were put onto these plates.
• The plates Incubated at 28°C for 24 hours in the dark.

4/3

• Switched spread plates to incubator at 36.8 for five days in the dark
• The images below are of the resulting plates.

4/8

• 100 µL of MP was spread onto minimal media plates and dried under a hood for two hours.
• 1 minimal media plate had 100 µL of PX spread onto the plate and dried under a hood for two hours.
  • The rest of the PX needed to be dried onto these plates at a later date due to a spill of PX which was properly cleaned.

4/9

• 100 µL of PX was spread onto the remaining minimal media plates.
• A streak of each bacteria from the spread plates was made on to these new plates.
• The plates were incubated at 36.8°C for six days in the dark
• The images below are of the resulting plates

04/16

• 100 µL of pesticide was spread onto minimal media plates and dried under a hood for two hours
• A toothpick of bacteria of the respective streak plates were put on to the grid plates based on the plate naming scheme.
• The plates Incubated at 36.8 for 3 days in the dark
• The images below are of the resulting plates.

04/19

• A toothpick of each bacteria on a grid was swirled into a tube with 100 µL of distilled H2O
• The plates were put in the fridge with tubes being put in the freezer so they can be prepared for sequencing at a later date.
• The image below has the tubes organized to match which grid location they were sampled from.

04/26

• The tubes were prepared to be sent out for sequencing using standard protocols. However, no gels were made to test the sequences quality before being sent.

04/28

• The tubes are sent out for sequencing.

04/29

• Sequencing results have been returned.
• Below are the sequences of each tube
  • AP EG 1
    • NNNNNNNNNNNNNNNGCGCAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGTGTTGTGGTTAATACCCGCNTCAATTGACGTTACCCGCAGAATAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCTGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCNAAACTGGCAGGCTAGAGTCTTGTANAGGGGGGTNNATTCCAGGTGTNNCGGTGAAATGCGTANNNANCTGGANGAATACCGNTGGCGAAAGGCGGCCCCCTGGACNNNNACTGACNCTCNNGGNGCGAAAGCGTGGGGNAGCAAAACAGGATTTAGATACCCCCGGTAGTCANA
  • AP EG 2
    • NNNNNNNNNNNNGGCGCAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGTGTTGTGGTTAATAACCTCAGCAATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCCNNGTAGTCANN
  • AP EG 3
    • NNNNNNNNNNNNNNNNNGGCGCAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGTGTTGAGGTTAATAACCTCAGCAATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCNNGTAGTCANN
  • AP EG 4
    • NNNNNNNNNNNNGGCGCAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGGGGAGGAAGGCGGTGAGGTTAATAACCTCATCGATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTCGAAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACNNCNNGTAGTCANA
  • AP EG 5
    • NNNNNNNGNNNNNNNNNGGNGCANCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGAGGAGGAAGGCGTTGTGGTTAATAACCTTAGCGATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTTGAAACTGGCAAGCTTGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCNCNNTANTCAAN
  • AP EG 6
    • NNNNNNNNNNNGGGCGNAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGAGGAGGAAGGCGTTAAGGTTAATAACCTTGGCGATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTCAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTTGAAACTGGCAAGCTTGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCCNNGNTAGTCAN
  • AP EG 7
    • NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
  • AP EG 8
    • NNNNNNNNNNNNNNNNNNNNNNCANNNNATGCACCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGNGNTAGGAAGGCGTTGTGGTTAATAACCTTGGCGATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTTGAAACTGGCAAGCTTGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCCAGTAGTCN
  • AP EG 9
    • NNGNNNNNNNNNNNNNNNNNCGCAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGTGTTGTGGTTAATACCCGGTTGAATTGACGTTACCCGCAGAATAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTACTCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTCTGTCAAGTCTGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTGNANACTGGCANGCTAGAGTCTTGTAGAGGGGGGTAGAATTTCNGGTGGTNCGGTGAAAATGCGTAGAGATCTGGNANNNNACNNNTGGCNAANGGCGNNNNCCTGGGACCNNCACTGACCNCTCNNGGNNCNAAAAGCGTGGGGGAGCNAAACAGGGATTTAGATNNCCCCNNGNAGTCANN
  • AP EG 10
    • NNNNNNNNNNNNNNNNNGGCGCAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGAGGAGGAAGGCGTTAAGGTTAATAACCTTGGCGATTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATTTGAAACTGGCAAGCTTGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCCNNGGTANTCA

05/03

• NCBI BLASTn was performed on 9 of the 10 sequences with the default settings.
• Tube AP EG 7 failed sequencing as it returned a sequence of 34 N in a row.

05/04

• Based on the results from BLASTn, conclusions of what genus the bacteria most likely classified as were made.

Sample Species AP EG 1 Enterobacter AP EG 2 Enterobacter cloacae AP EG 3 Enterobacter AP EG 4 Klebsiella variicola AP EG 5 Raoultella planticola AP EG 6 Klebsiella sp. DB2 AP EG 7 Failed sequencing AP EG 8 Klebsiella sp. DB2 AP EG 9 Enterobacter AP EG 10 Klebsiella sp. DB2