Construct 2: Performed by Vishwaa K., Edward K., Leon G.

• Competent cells were thawed for 25 minutes on ice.

• Mixed 0.5 µL plasmid with 1.5 µL water in 1.5 mL DNA tube.

• Vortex the diluted plasmid DNA for 2 seconds.

  • Note: Edward Centrifuged for too long (20 seconds).

• Added 50 µL E. coli to DNA tube.

  • Note: Leon left some of the mix of the 1.5 mL tube in the pipette tip.

• Flicked the 1.5 mL DNA tube to mix the DNA and competent cells gently.

• Placed the tube on ice for 25 min.

• Resealed the plasmid storage tubes and returned them to the freezer.

• Heat shocked the transformation tube at 42°C for 30 seconds.

• Placed heat-shocked 1.5 mL DNA tube on ice for 2 minutes.

• Added 500 µL of SOC media to the transformation reaction.

• Placed the transformation tube on a shaker at 200 RPM and 37°C for 60 min.

• Pipetted 100 µL X-Gal and 40 µL IPTG on six kanamycin plates: 3 high-concentration (100 μg/mL) plates and two low-concentration (50 μg/mL) plates.

• Spread the X-GAL and IPTG evenly on each plate and let the plates dry.

• Plates were closed with lids until the bacteria were done shaking.

• Added bacteria to plates; plates had the following µL of bacteria, respectively:

  • High-concentration plates: 150 µL, 100 µL, 50 µL.

  • Low-concentration plates: 30 µL, 10 µL.

• Incubated overnight at 37°C.

Construct 1: Performed by Shlok J., Akhila N., James M.

• Thawed E. coli bacteria on ice for approximately 25 minutes.

• Thawed plasmid DNA (containing prma promoter) and vortexed briefly.

• Added 1.5 µL of water into a separate microcentrifuge tube.

• Pipetted 0.5 µL of DNA (4 ng/mL) into the water tube to prepare the dilution mix.

• Vortexed the DNA dilution mix again.

• Placed the DNA solution back on ice.

• Added 50 µL of E. coli into the microcentrifuge tube and returned it to ice.

• Incubated the E. coli/DNA mixture on ice for 25 min.

• After the incubation, the ice bucket was taken to the water bath, and the E. coli/DNA tube was heat-shocked for 30 sec at 42°C.

• Returned the E. coli/DNA tube to ice for 2 min.

• Added 500 µL of SOC media to the bacteria tubes and incubated them in a shaking incubator.

• Incubated the tubes for 60 min at 37°C and 225 rpm.

• Waited 2 mins to put on shaking incubator with Construct 2.

• Pipetted 10 µL and 20 µL E. coli/DNA mixture onto 10cm LB selective agar plates.

• Gently spread bacteria throughout agar plate.

• Incubate plates at 37°C overnight (putting plates upside down).

Selection for Construct 2 - 7/11/2024 Performed by Vishwaa K.

• Opened plates in the fume hood @ 8:30 AM the next day

• Found good growth in Construct 2 plates

• Placed in a 4°C fridge so that blue colonies can emerge

  • Note: The plate with 10 µL of bacteria worked best

• Circled white colonies on all plates with Sharpie on the back of the plate

• Poured 50 mL of LB broth into a tube

• Added 500 µL of Kanamycin to the tube & mixed by inverting

• Aliquoted 5 mL of the solution in each of 10 cell culture tubes under the fume hood

• Used Sterile Pipette tips to select a colony

  • Swiped colony with the sterile end of the pipette tip

  • Placed full pipette tip inside cell culture tube

  • After inserting the pipette tip, the cell culture tubes were closed halfway to allow the cells to breathe.

• Placed the tubes in the incubator at 225 rpm, 37 °C overnight

• Plates were sealed with parafilm and stored at 4 °C

Selection for Construct 3 7-12-24 - Aryan S, Gowshik R, Daniel J

Circled white colonies on all plates with Sharpie on the back of the plate

• Poured 50mL of LB broth in a tube

• Added 500 µL of Kanamycin to the tube & mixed by inverting

• Aliquoted 5mL of the solution in each of 10 cell culture tubes under the fume hood

• Used Sterile Pipette tips to select a colony

  • Swiped colony with the sterile end of the pipette tip

  • Placed full pipette tip inside cell culture tube

  • After inserting the pipette tip, the cell culture tubes were closed halfway to allow the cells to breathe

• Placed the tubes in the incubator at 225 rpm, 37 °C overnight

• Returned plates to 4 °C fridge after properly sealed with parafilm

* Old negative controls thrown out

• Added 25 mL LB Broth in broth-Kanamycin solution tube

• Added 250 µL Ampicillin and 250 µL Kanamycin into the tube containing 25 mL LB broth

  • Mixed by inverting

• Aliquoted 5 mL of the solution to the tubes

• Circled positive colonies for the redo plates - 7/12/24

Construct 1

Due to other members not doing their job, proper notes of what happened were not kept. But the procedures (in wetlab experiments page) contain the steps followed.

Miniprep of Construct 2 - Performed by Vishwaa K., Leon G., and Edward K. - 7/12/2024:

• Removed cell culture tubes at 9:50 AM from the incubator

  • Note: Leon dropped our cell culture tubes, but none spilled

• Used a miniprep kit (opened but unused)

• Aliquoted 1.5mL of each colony into a respectively labeled microcentrifuge tube

  • Meaning they were labeled Colony 1 - Colony 5

• Centrifuged at 8000 rpm and room temperature for 2 mins

• Labeled five more 1.5mL microcentrifuge tubes (Colony 1-Colony 5) for storage

• Removed supernatant of microcentrifuge tube

• Added 250 µL of Resuspension solution to each microcentrifuge tube

• Flicked tubes gently until cell clumps completely dissolved

• Added 250 µL of Lysis Solution to each tube and inverted 6 times to mix evenly

• Added 350 µL of the Neutralization Solution and mixed by inversion until cloudy

• Centrifuged the tubes at 12000 rpm and room temperature for 5 min

• Took 650 µL of each microcentrifuge tube’s supernatant and placed it into their respectively labeled spin column

• Centrifuged the spin columns at 12000 rpm and room temperature for 1 min

• Repeated twice

  • Discarded flow through of each spin column

  • Added 500 µL wash solution to each spin column

  • Centrifuged the spin columns at 12000 rpm for 1 min

• Discarded the flow-through of each spin column

• Centrifuged all the spin columns at 12000 RPM for 1 min

• Transferred the filter part of each spin column to their respectively labeled microcentrifuge tube

• Added 50 µL of elution buffer to the center of each spin column filter

• Incubated at room temperature for 2 minutes

• Centrifuged at 12000 RPM for 2 min

• Discarded filter columns and placed the microcentrifuge tubes with the flow through on ice

• Nanodropped samples for each of the five colonies

Construct 1: Miniprep

Due to other members not doing their job, proper notes of what happened were not kept. But the procedures (in wetlab experiments page) contain the steps followed.

Construct 3: Miniprep

Due to other members not doing their job, proper notes of what happened were not kept. But the procedures (in wetlab experiments page) contain the steps followed.

Digest of Construct 2 - Performed by Vishwaa K., Leon G., and Edward K. - 7/12/2024:

• Pipetted a master mix of 5 µL Eco31i, 25 µL G-buffer, and 120 µL water in a 1.5 mL microcentrifuge tubes

  • Gently Vortexed and Mixed

• Created microcentrifuge tubes with a mix of plasmid DNA and water, equaling 20 µL

  • See after nanodrop results for exact measurements

• Added 30 µL of master mix to each of the 5 microcentrifuge tubes

• Incubated the five tubes at 37 °C for 1.5 hours

• Moved the five microcentrifuge tubes of plasmid DNA to 4 °C for overnight storage and stored purified DNA at -20 °C

Repoured plates for construct 3

• Removed colonies from incubating overnight and placed at 4 °C after incubating overnight

Construct 1: Digest

Due to other members not doing their job, proper notes of what happened were not kept. But the procedures (in wetlab experiments page) contain the steps followed.

Construct 3: Digest

Due to other members not doing their job, proper notes of what happened were not kept. But the procedures (in wetlab experiments page) contain the steps followed.

Construct 2: Nanodrop Results

Construct 1: Nanodrop Results

Due to other members not doing their job, proper notes of what happened were not kept. But the procedures (in wetlab experiments page) contain the steps followed.

Construct 3: Nanodrop Results

Due to other members not doing their job, proper notes of what happened were not kept. But the procedures (in wetlab experiments page) contain the steps followed.

Calculations for 1μg (Digest) for Construct 2

Nanodrop Results

Restriction Digest

• Labeled 18 tubes for digest

  • Red: Construct 1

  • Green: Construct

  • Black: TF

  • Orange: Gene

  • Blue: Construct 3

(All microliters of sample + dH2O)

• Made digest mix

  • 90 µL of buffer G

  • 18 µL of enzyme EcoRI

  • 432 µL of H2O

  • Vortexed and spun down

  • Added 30 µL of digest mix to each tube of 20 µL (plasmid DNA and water)

  • Vortexed, spun down, and incubated at 37.0 degrees Celsius for 1.5 hours

Plating colonies

• Construct 1 - prmA

  • Colony 4 Kan

  • Colony 5 Kan

• Construct 2 - FAB

  • Colony 2 Kan

  • Colony 3 Kan

• Construct 3 - Estradiol

  • Colony 5 (More TF) Both

  • Colony 1 (More gene) Both

• Construct 3 - TF

  • Colony 1 Kan

  • Colony 2 Kan

• Construct 3 - Gene (No insert, probably)

  • Colony 1 Amp

  • Colony 2 Amp

• Labeled 10 culture tubes for the plate

• Four were smaller because we ran out of large cell culture tubes (2 colonies per construct + 4 negative controls)

• Made stock for Kan, Amp, and Both (All mixed by inverting)

  • Kan

    • 35 µL LB Broth + 350 µL Kan (100x)

  • Amp

    • 15 µL LB Broth + 150 µL Amp (100x)

  • Both

    • 15 µL LB Broth + 150 µL Amp (100x) + 150 µL Kan (100x)

• Pipetted 5 mL or 3 mL to each tube respective of size and antibiotic

• Pipetted 5 µL or 3 µL of original media to new media respective of size and antibiotic

• Opened lid slightly

• Incubated overnight at 37 degrees Celsius and at 210 RPM

Adding PFOA and H2O2

• Labeled 3 tubes for selective LB Broth

  • 30 mL Kan (29.7 mL broth + 300 µL Kan (100X))

  • 10 mL Amp (9.9 mL broth + 100 µL Amp (100X))

  • 15 mL both (14.7 mL broth + 150 µL Kan (100X) + 150 µL Amp (100X))

• PFOA Dilutions

  • 1 mL stock A_p: 875 µL LB Broth + 125 µL of 20 mM PFOA = 2500 µM PFOA

  • 1 mL stock B_p: 800 µL LB Broth + 200 µL of stock A_p = 500 µM PFOA

  • 1 mL stock C_p: 900 µL LB Broth + 100 µL of stock B_p = 50 µM PFOA

• H2O2 Dilutions

  • 10 mL stock A_h: 9997.45 mL LB Broth + 2.55 µL 30% H2O2 = 2500 µM H2O2

  • 1 mL stock B_h: 800 µL LB Broth + 200 µL of stock A_h = 500 µM H2O2

  • 1 mL stock C_h: 900 µL LB Broth + 100 µL of stock B_h = 50 µM H2O2

• Overnight culture + nanodrop the following day:

Note: Letters are out of order due to a labeling mistake, but now they are correct.

Testing fluorescence

• Aliquots:

• Added PFOA to all wells

• Read plate for OD + fluorescence (results in google sheets)

• T2 Plate 2 reading ‘Too saturated’