Experiments

1. Aliquot out 1-2 µL of DNA into microcentrifuge tubes. (This can be a PCR product, or Miniprep. Must be circularized)
2. Take out competent cells from -80. Let thaw on ice for 30 mins. (If BL21 cells, thaw for 10 mins)
3. Set the water bath to 42 degrees Celsius.
4. Warm selection plates in the incubater at 37 degrees Celsius.
5. Once cells have thawed, add 25 µL of cells into each microcentrifuge tube.
6. Mix gently (flicking, no vortexing), and let sit for 30 minutes on ice.
7. Heat shock cells for 30s. (If BL21 cells, heat shock for 10s)
8. Put cells immediately back on ice for 5 mins.
9. Add 475 µL outgrowth medium to the cells.
10. Let incubate at 37 degrees Celsius and shake at 400 rpm for 1 or more hours.
11. Plate 50-100 µL of outgrowth solution onto the selection plate.
12. Spread on plate or serial streak.
13. Let incubate overnight.

1. Take out electroporation cuvettes and place on ice for at least 10 minutes.
2. Aliquot out 1-2 µL of DNA into microcentrifuge tubes. (This can be a PCR product, or Miniprep. Must be circularized.) IMPORTANT: Make sure DNA is suspended in Nuclease-Free Water or a TE solution with minimal salts. If not, could cause potential harm to users.
3. Take out competent cells from -80. Let thaw on ice for 10 mins. Confirm they are thawed by gently flicking the tube.
4. Warm selection plates in the incubater at 37 degrees Celsius.
5. Add 25µL(1) competent cells to the DNA tubes, and gently flick to confirm they are mixed. Be careful to not take out cells from ice for too long
6. Load the DNA/Cell mixture inside the gap of the cuvette. Make sure there are no air bubbles above the cells
7. Turn on the electropulser. Set settings as specified by your cuvettes.
8. Pulse the electropulser, and quickly add 975µL(1) of SOC or Outgrowth Media
9. Pippette up and down in the cuvette, and transfer 1000µL to a separate centrifuge tube
10. Let incubate at 37 degrees Celsius and shake at 400 rpm for 1 or more hours.
11. Plate 50-100 µL of outgrowth solution onto the selection plate.
12. Spread on plate or serial streak.
13. Let incubate overnight.

1: More cells may be used based on cuvette specifications; depends on the supplier and gap width. Amount of outgrowth is calculated as: 1ml - (amount of cells)

1. From selection plate, circle one or more isolated colonies.(1) Number them or mark them to distinguish between them.
2. Take out the same number of culture tubes as colonies.
3. Set aside two tubes; these will serve as controls. To each tube, add 3mL of the associated antibiotic in LB. Click here for the protocol
4. To the set aside tubes, follow these steps:
   1. This one is the positive control. To the tube, add only 3mL of LB only- no antibiotic
   2. This one is the negative control. To the tube, add 3mL of LB that contains a separate antibiotic not possessed by the bacteria
5. Now, number the tubes based on the colony on the plate
6. Using either a toothpick or a pipette tip (assuredly sterile), lightly tap the colony and then quickly place the tip/toothpick in the culture tube corresponding to the mark.
7. After this has been repeated with all the colonies, place the tubes in an incubating shaker.
8. Let the tubes incubate at 37 degrees Celsius for 12-16 hours or at 30 degrees Celsius overnight.

1: This procedure may be altered to save time; if one is confident about their cells, they can move directly from the outgrowth step of the transformation step.

1. From liquid culture, pellet ~1.5 mL of cells in media. Centrifuge at 13000rpm for 1 min.(1)
2. Discard supernatant. Resuspend in 200µL of Buffer 1(Plasmid Resuspention buffer). Invert until the pellet is resuspended, vortex as needed.
3. Add 200µL of Buffer 2(Plasmid Lysis Buffer), gently invert 5-6 times and incubate at room temperature for 1 minute.
4. Add 400µL of Buffer 3(Plasmid Neutralization Buffer), gently invert until neutralized. When the liquid turns yellow, let it incubate at room temperature for 2 minutes
5. Centrifuge the liquid for 2-5 minutes at 13000 rpm.
6. Set up a spin column, labelled with the plasmid of choice.
7. Carefully pour the supernatant into the column and centrifuge at 13000rpm for 1 minute.
8. Discard supernatant. Re-insert column and load 200µL of Wash buffer 1. Spin at 13000rpm for 1 minute.
9. Discard supernatant. Re-insert column and add 400µL of Wash buffer 2. Spin at 13000rpm for 1 minute.
10. Transfer column carefully to a clean centrifuge tube
11. Carefully add in 20-30µL of Elution buffer (TE or NFW). Make sure to load directly onto the column.
12. For best results, incubate at 37 degrees celsius for 5 minutes before the next step.(2)
13. Spin at 13000rpm for 1 minute. Discard the column.

1. For this step OD must not exceed 15 units.
2. For the best yield, heat up elution buffer to 50 degrees celsius before this step.

1. To begin, we must have a liquid culture with OD600 > 0.4.
2. Pipette up and down until the solution is turbid. Aliquot out ~1mL of the liquid culture into a microcentrifuge tube.
3. In the tube, add glycerol until the final concentration is about 20%.
   1. Given 1mL starting concentration, add around 666µL of 50% glycerol stock, or 333µL of 80% glycerol.
4. Gradually lower the temperature of the cells:
   1. First place the cells on ice for 10 minutes
   2. Now, place the cells at 20 degrees celsius for 10 minutes
   3. Finally, place the cells in the -80. Stores up to 6 months.

1. Begin with a 3mL overnight culture of the necessary cells. Ensure the plasmid and any constructs are present.
   1. Prepare 400+mL of autoclaved DI water, and store in the fridge
   2. Prepare a solution of autoclaved 80% glycerol
2. After the culture has reached OD600 of greater than 0.5, aliquot out 1mL of culture.
3. Prepare an erlenmeyer flask with 100mL of LB with the associated antibiotic resistance. Add in the 1mL aliquot.
4. Incubate again at 37 degrees celsius for 4-6 hours, until OD600 reaches 0.5-1.
5. Take the flask out, and immediately chill on ice for ~30 minutes.
6. From the flask, aliquot 50mL and place in a chilled falcon tube. Harvest cells by centrifugation at 3200rpm at 4 degrees celsius for 20 minutes
7. After discarding the supernatant, resuspend the pellet in 50mL of cold DI water.
8. Spin the cells at 3200rpm at 4 degrees celsius for 20 minutes again.
9. After discarding the supernatant again, resuspend the pellet in 25mL of cold DI water.
10. Spin the cells at 3200rpm at 4 degrees celsius for 20 minutes again.
11. Discard the supernatant. To each tube, add in 21.875mL of ice-cold DI water, and add in 6.25mL of ice-cold glycerol.
12. Make sure the tube is well mixed. Dispense out 100µL aliquots of the stock, and freeze immediately.
    1. In a styrofoam box, prepare some dry ice. When close to freezing time, add in some ethanol to create a slush
13. Once done dispensing, immediately transport tubes for storage in the -80 degrees celsius.
14. Cells prepared in this manned are best used in 1mm cuvettes, at 2500V, 200Ω, and 25 µF.

1. Design the KO Inserts, as well as two sets of primers to amplify each insert.
2. After ordering the inserts, and ensuring that all features are complete, begin by amplifying the DNA ordered. When targetting promoter regions, the key features to ensure are:
   1. 600+bp recombination site. Should be just downstream of either the promoter or the UTR.
   2. Antibiotic resistance or other selection method: Best way to ensure successes without needing to genomically sequence
   3. Two constitutive terminators: Should be upstream of the recombination site, and downstrem from the antibiotic resistance
   4. Constitutive promoter: Should be upstream of the antibiotic resistance. Ensure it faces the right direction, and contains an RBS.
3. Set up PCR amplification reactions with each insert, and then run for 30+ cycles.
4. Run a portion of the PCR result on a gel (3-4 µL).
   1. If only one band is present, PCR cleanup or ethanol precipitate to remove residual salts.
   2. If multiple bands are present, create a second gel, with the purpose of gel purification at the desired kb count.
5. Now, use a nanodrop to determine the amount of DNA present.
6. Set up two reactions (Assembly and MegaWhop), both with a 1:9 molar ratio in backbone to insert. If the two constructs are the same length, choose one arbitrarily to be the backbone.
7. After the reactions have occured, run a second ligation step on the reactions, to ensure that all nicks are repaired(1)
8. Run the result on a gel to ensure that the expected band is seen. Circularized DNA runs a little faster than linear DNA, so the band should be lower than expected.
9. Gel purify the result, regardless of whether there are multiple bands or just one band.
10. In the elution step, ensure that Water or TE is used.
11. For best results, perform an outgrowth step of 3 hours, and transform using electroporation.

1: Because the "plasmid" has no origin of replication, the Bacterial DNA repair cannot recognize it as a PCR. Therefore, we must avoid leaving nicks, as they will make recombination messy.

The usage of in-house materials to recreate an anaerobic chamber and assess metabolic efficiency. The anaerobic chamber set up involved a two-part plastic chamber with a space separated by a grate where O₂ absorber packs were placed with a dynamic O₂ indicator. In the presence of O₂ the indicator is a dark-blue color, and in an anaerobic environment, the indicator turns red.

Anaerobic experiment 1

Objective: Observe G4 ecoli growth on M9 minimal media supplemented with 150uL of 10mM saponified hexanoic acid in anaerobic and aerobic environments.

Setup: Normalizing the colonies plated

1. Glycerol stocks of G4 and wildtype were serial streaked on an ampicillin plate and incubated at 37C overnight.
2. Isolated colonies were picked the next day and labeled G4A/B/C and WTA/B.
3. Liquid cultures were made using the picked colonies in 3mL of ampicillin LB(G4) and 3mL of plain LB(WT).
4. The cultures were grown for 12 hours and OD600 measurements were taken.
5. Cultures were then normalized to obtain a standard OD of 0.710, the lowest OD.
6. These cultures were then diluted to a factor of 10^(-1 through -4) by pipetting 100uL of the previous culture into 900uL of ampicillin or LB broth. This process was repeated until the max dilution of 1: 10^-4 was obtained. 25uL of each serial dilution was then plated on ampicillin LB agar or on plain LB agar
7. After 24 hours of growth, colonies were counted on the most dilute plate to determine the original number of CFUs plated.
8. G4A x 10^-4 had 256 individual colonies; G4B x 10^-4 had 603; G4C x 10^-4 had 547; WTA had 1258 and WTB had 2212. CFUs were calculated by taking the number of colonies, and dividing by the mL of solution plated times the dilution factor. G4A had 1.02E8, G4B had 2.41E8, G4C had 2.19E8, WTA had 5.03E8, and WTB had 8.85E8 CFUs/mL This number is then used to determine the starting CFUs plated on each.

Aerobic and Anaerobic setup

• 5 technical replicates were used G4A/B/C/E1/E2
• 2 replicates of WT were used WTA/B
• 4 blue control plates with nothing on them were used to collect baseline blue levels
• 8 O2 reducer packet
• 2 O2 pink/blue indicators

Over the course of two trials, all replicates were grown in aerobic and anaerobic conditions.

Trial 1:

• 30uL of each G4A/B/C were plated on LB with Ampicillin (3 plates)
• 30uL of each WTA/B were plated on LB only (2 plates)
• 4 each of 30uL of G4 and WT were plated on 150uL of 10mM saponified hexanoic acid on M9 minimal media (20 plates) for a total of 25 plates anaerobic/aerobic

The same number of plates were tested anaerobically and aerobically at the same time.

The 25 plates were stacked into the anaerobic jar, the seal rubbed with petroleum jelly, and then through a three-way stopcock, N2 gas was pumped into the jar while the O2 left. Then, a slight vacuum was applied to the container, stopcock closed, and placed in the incubator for 5 days at 37C.

The 25 aerobic plates were placed directly after plating into the incubator for 5 days.

Observations

Trail 1 D1: there was no visible growth on the plates, this was expected, as we had tried growing G4 on Hexanoic acid minimal media aerobically, and did not see any changes to the plate for around 3 days. Aerobic LB and ampicillin plates have considerable growth.

Trial 1 D2, Aerobic plates had small pinpoint size potential colonies of growth. Anaerobic was difficult to see inside the jar.

Trial 1 D3: More small specks on other plates, but no change to pre-existing specks.

Trial 1 D4: Aerobic plates did not have distinct colonies, but a thin layer of potential growth.

Trial 1 D5: Jar was opened at 10:00am for a total of 4d 15 hr of growth. Almost all plates exhibited the same sheen of growth that aerobic counterparts had.

Conclusion: More trials were needed to create a conclusive argument. Decided on a second trial testing media that had the inclusion of 20mM NO3, which according to some literature, the lack of nitrate in the plate prevents e.coli from growing anaerobically. This led to the design of trial 2.

Trial 2:

G4B/G4C/G4E1/2 and WT A/B were replated from glycerol stock, allowed to grow for 24 hours on LB ampicillin agar (plain LB for WT). From this, colonies were picked from each and liquid cultured using respective liquid media. The same procedure as before was used to normalize the starting OD. For this trial, the M9 minimal media was prepared with Ca(NO3)2, the m-salt solution was prepared and Ca(NO3)2 was added to a 10mM concentration. The process of adding the calcium nitrate to the m-salt solution forming a white precipitate that was the Ca binding to the PO4 ions from the NaPO4 creating. This precipitate was then filtered out of solution, leaving behind 20mM of NO3 which was then added to the M9 Minimal media solution before adding the agar and autoclaving. This trial also introduced Glycerol/Amp on M9 minimal media. To prepare these plates, 50mL of the M9 solution with agar was autoclaved, once cool 1uL of Amp100 was added per mL of solution. Due to the space available(25 plate max) in the anaerobic chamber the number of Plates of G4B/C/E1/E2 on M9 with Nitrate was reduced to 3 each(12)

• 1 ea G4B/C/E1/E2 of M9 with glycerol/amp(4)
• 1 ea WTA/B on M9 with glycerol- no antibiotic (2)
• 1 ea G4B/C/E1/E2 on LB/amp plates(4)
• 1 ea WTA/B on LB only (2)

Due to the time constraint of iGEM wiki freeze, Trial 2 was left to grow for three days.

1. Measure out 20 mL of TBE (Tris-borate-EDTA) buffer.
2. For a 1% gel, measure out 0.2g of Agarose. For a 2% gel, measure out 0.4g agarose.(1)
3. Combine the agarose and TBE, and then microwave for 30s. Add 10s as needed, until solution is clear and all agarose is dissolved.
4. Let the solution cool for around 10 minutes(2). When it has cooled a bit, add 2.5-3 µL of CybrSafe(invitrogen).
5. After the container the gel is in is cool enough to hold, but still liquid, pour the gel into the mold. Make sure to cast with a comb as well.
6. Allow the gel to set. In the meantime, add loading dye to your DNA. The formula is: µL DNA/4 = Dye needed to be added.
7. After the gel has set, move the gel into the gel electrophoresis chamber. Make sure that the diodes are unoccluded.
8. Into the chamber, pour in extra TBE as needed, such that the gel is fully submerged. (3)
9. Into the first well of the gel, load the corresponding ladder needed. The formula is as follows: (µL DNA + dye)/4 = µL DNA.
10. Load the DNA into each well.
11. Run first at 50V for 1 minute, then 135V for 25 minutes. If not possible, just run at 135V for 25 minutes.
12. Document the results of your gel.

1. If a larger gel is needed, the formula largely stays constant. Use the ratio to determine the amount of agar and TBE needed.
2. For 2% gels, the cooling time cannot occur, and the gel should add the cybrsafe as soon as possible.
3. When running a gel for the purpose of gel purification, make sure that all TBE in the chamber is fresh.

1. Excise the band wanted from the gel. Use a scalpel to first cut along the well of the gel, and then cut around the band. Make sure to limit gel exposure to UV light.
2. After the gel has been cut, store into a microcentrifuge tube, and then set aside.
3. Measure the mass of gel that has been cut. Then, add 4x the volume of Gel Dissolving buffer. (for every 100mg of agar, 400µL of buffer should be added)
4. Incubate at 50 degrees celsius, and shake at 500rpm for 7-10 minutes.
5. After the gel has fully dissolved, load the solution into a spin column.
6. Spin at 13000rpm for 1 minute. Discard the supernatant.
7. Re-insert the spin column, and load 200µL of Wash Buffer.
8. Spin at 13000rpm for 1 minute. Discard the supernatant.
9. Repeat the last two steps.
10. Add 5-6µL of DNA Elution buffer (TE)
11. Incubate at 37 degrees celsius for 5-6 minutes.
12. Spin at 13000rpm for 1 minute. The supernatant is the DNA.

1. Identify the colonies of interest, and circle them on the plate.
2. Create a mastermix using 10µL of OneTaq, 10mM of each primer of interest, and any NFW that may be needed
3. In the number of PCR tubes corresponding to the number of colonies, add in the mastermix.
4. Pick each colony with a toothpick, and then quickly swirl them around in the PCR tube.
5. Let the toothpicks sit in solution for a bit. Set up liquid cultures for the colonies picked.
6. Set up the thermocycler with a melting temperature of 98 degrees Celsius, and a melting time of 2 minutes initially.
7. For each cycle, the melting time should be 1 minute at 98 degrees Celsius, and the annealing time and temperature should be 30s and the same as the annealing temperature of the primers.
8. The extension time should be calculated as follows: length of 30*(insert(kb)/1000)
9. Let this run for 30 cycles. Then, run the solution on a gel.

1. Calculate the fold excess of each fragment using this spreadsheet.
2. Dilute the fragments as needed.
3. Into a PCR tube, add 10µL of Assembly master mix, along with the necessary NF Water.
4. Add in the DNA, and set up the Thermocycler. Based on the master mix, set the temperature as needed. For us, we set it to 50 degrees celsius for 15 minutes.
5. After the reaction is over, continue with the PCR product as needed.

1. Calculate the molar ratios of each fragment. For reference, the molar ratio for backbone to insert is best at 10fm to 20fm.
2. Dilute the DNA to the required amounts. Begin thawing T4 Ligase Buffer
3. Using the number of reactions as a metric, calculate the total amount of T4, T4 Buffer Enzyme, Enzyme Buffer, and NF Water needed
4. Aliquot the appropriate amount of master mix to each PCR tube
5. Add in the Backbone and insert
6. In the PCR, set up a 4-step reaction with an initial temperature of 37 degrees celsius for 2 minutes.
7. Then, the second temperature should be set at 16 degrees celsius for 5 minutes, run for 20-50 cycles. 20 cycles for quick results, 50 cycles for best results.
8. Then, set the remaining two temperatures to the enzyme's inactivation temperature, for 10 minutes, and the ligase's inactivation temperature for 10 minutes.
9. After the reaction is over, transform for the best results

1. Ensure that the megaprimer will anneal to the backbone, and find the exact annealing temperature and associated amplification length.
2. In a PCR tube, add 12.5µL of Q5 MM, and any necessary NF Water.
3. A fold ratio of at least 1:9 is recommended from backbone to megaprimer. After this ratio has been achieved, add both to the PCR tube.
4. Run the Thermocycler at a melting temperature of 98 degrees celsius, and an annealing temperature of 5 under that displayed on benchling.
5. Run for at least 30 cycles. Then, for the best results, directly DPNI digest and transform.

1. Familiarize yourself with the enzyme's activity; Optimal temperature, inactivation temperature, etc.
2. To the DNA you wish to digest, add 1µL of the enzyme, and then any buffers that may be necessary.
3. Now, incubate the enzyme at the most active temperature for 1-3 hours.
4. To test if the digest was successful, run some on a gel as needed. (For DPNI, this is unnecessary)

1. Firstly, make sure to have designed primers that face eachother, and amplify the wanted insert.
2. Find the annealing temperature on benchling. Then, calculate the average if needed.
3. In a PCR tube, add in 12.5 µL of Q5 Master Mix, along with the necessary NF Water
4. Add in 10mM of each primer, and your insert. Tune the thermocycler as necessary
5. You may run 30-50 cycles, depending on need. Be sure to run the results on a gel to determine if a gel purification is needed.

1. Measure out the following: 400mL of DI water, 6g Luria Broth Powder.
2. In the bottle, which must be at least 500mL in total capacity, add in the Luria Broth Powder, and mix until the liquid is no longer clear.
3. Ensure the bottle's cap is slightly open, and then place one piece of autoclave tape over the cap.
4. Bring the bottle to the autoclave. In the chamber, add some water to the bottom of the container the bottle is in to ensure that any spillage will be easy to clean. Now, run a L30 cycle.
5. After the bottle has been autoclaved, carefullt remove it from the hot chamber.
6. Allow the media to cool, and be sure to note that the autoclave tape has changed color.
7. After the bottle has cooled, screw the cap on tight, and store indefinitely on benchtop.
8. If antibiotic resistance is needed, add in the corresponding amount of µL of Antibiotic to the amount of mL of media. (e.g. 400mL LB = 400µL Kanamycin-15 needed to be added)
9. Use as needed.

1. Measure out the following: 400mL of DI water, 6g Luria Broth Powder.
2. In the bottle, which must be at least 500mL in total capacity, add in the Luria Broth Powder, and mix until the liquid is no longer clear.
3. Ensure the bottle's cap is slightly open, and then place one piece of autoclave tape over the cap.
4. Bring the bottle to the autoclave. In the chamber, add some water to the bottom of the container the bottle is in to ensure that any spillage will be easy to clean. Now, run a L30 cycle.
5. After the bottle has been autoclaved, carefullt remove it from the hot chamber.
6. Allow the media to cool, and be sure to note that the autoclave tape has changed color.
7. If antibiotic resistance is needed, add in the corresponding amount of µL of Antibiotic to the amount of mL of media. (e.g. 400mL LB = 400µL Kanamycin-15 needed to be added)
8. Once the bottle is cool enough to touch, take out a fresh sleeve of untouched plates, and pour media as needed.
9. Allow plates to cool on benchtop overnight or open in a flow hood for ~3 hours.
10. Once condensation is gone, place plates in the 4 degrees Celsius Fridge.
11. Use as needed.

1. Measure out 12 g of agar and 300mL of DI water. Mix the solution together
2. Prepare 500mL of MSalt solution:
   1. Measure out 13.8g of Na2HPO4. Add to a 500mL glass bottle.
   2. Measure out 3g of KH2PO4. Add to the same bottle.
   3. Measure out 1g of NH4Cl. Add to the same bottle.
   4. Measure out 0.5 of Na2SO4. Add to the same bottle.
   5. Fill the bottle to 500mL with DI Water.
3. Ensure the bottles' caps are slightly open, and place autoclave tape over the caps.
4. Bring the bottles to the autoclave. In the chamber, add some water to the bottom of the container the bottle is in to ensure that any spillage will be easy to clean. Now, run a L30 cycle.
5. Once the bottles have cooled enough, mix the two solutions in a 1:1 ratio.
6. Allow the bottles to cool to < 60 degrees celsius. Add in 6mL of ATCC Trace mineral supplement and antibiotic if needed. Also add 2.25mL trypan blue for more visibility of plates if needed.
7. Once All materials have been mixed, quickly pull out a sleeve of plates and pour the media.
8. Allow plates to cool on benchtop overnight or open in a flow hood for ~3 hours.
9. Once condensation is gone, place plates in the 4 degrees Celsius Fridge.
10. Use as needed.

1. Calibrate nano pH meter.
1. Rinse probe with DI water.
2. With standard 4.00pH buffer, place probe in liquid and allow meter to calibrate to 4.00pH.
3. Repeat with 10.00pH buffer and 7.00pH buffer, being sure to rinse with DI water between buffer solutions.
4. Note: Do not allow probe tip to dry out, have storage buffer solution on hand.
2. In a 100mL beaker, add 25mL of DI water.
3. Add 31.3 µL of Hexanoic acid or 2,2 -difluorohexanoic acid.
4. Add 1 µL of M-Salt solutions as a buffer.
5. Record the initial pH.
6. Add stir bar, set stir plate to low speed
7. Add 0.1M NaOH in 25µL increments until pH is ~7.
8. Add 1% of total volume as NP-40 detergent, stir for roughly 30s to 1m.
9. Pour liquid in sealable falcon tube and label.

1. After the plates have exhibited the growth for the period, count the colonies present either manually or using a script.
2. Calculate the CFU's using the formula: (colonies)/(milliliters plated * serial dilution)
3. An easy link is this spreadsheet here

1. Once plates are ready for data collection, they were placed in a custom made photo box that allowed for consistent images.
2. Images were uploaded to Google Drive and then processed using (Fiji is just)ImageJ.
3. A circular region of interest(ROI) was selected on the blue control plate (no cells) being sure to include as much of the plate as possible without any label or external markings.
4. Pressing ctrl+h brings up a histogram analyzing the color channels present in the ROI.
5. Cycling through the histogram, the blue channel is selected and the mean level is recorded for each plate on a spreadsheet for future analysis.
6. For subsequent images, press ctrl+shift+e to restore the previous ROI onto the new image. This ensures consistency in the ROI from image to image.
7. To normalize the data, a control plate was grown overnight at the same time as the experiment start on ampicillin media, and then contrast was done by (control mean blue - test condition mean blue)/(control mean blue)

Data Analysis