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Description Design Experiments Notebook Contribution Results Demonstrate Improve Attributions
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Basic Parts
Safety (current)
Human Practices
Human Practices
Awards (current)
Judging Form (current)

Experiments

Materials
1. Agar
2. Antibiotic
3. Empty plate
Procedure
1. Heat the agar for 15 minutes at power 4 in the microwave or until melted
2. Retrieve empty plates and label with correct antibiotic
3. Retrieve antibiotics and keep in ice, allow them to defrost
4. Sterilize the area with ethanol/water mixture, and prepare a flame
5. Add antibiotic to the container of agar
  1. 1000:1 ratio of agar:antibiotic
6. Flame containers and pour agar into plates
  1. Just enough to cover bottom (around 5mL)
7. Remove any bubbles with flame
8. Let cool and solidify, then place in fridge

Materials
1. DNA Plasmid
2. Plate with correct antibiotic
3. Culture beads
4. 37 °C incubator
5. 37 °C shaker
6. Hot water bath
7. Ice bath
8. LB media
9. Microcentrifuge tubes
10. GeneHogs cells
Procedure
1. Retrieve GeneHogs cells in ice bucket and allow them to thaw
2. Resuspend DNA plasmid by adding 10μL of autoclaved nanowater to the correct well of the appropriate plate and then let sit for 5 minutes
3. Prepare the heat-block water bath by filling a couple holes with water
4. Sterilize the area and instruments with ethanol/water mixture, and prepare a flame
5. Retrieve microcentrifuge tube and label them accordingly
6. Transfer 50μL of GeneHogs and 1μL of DNA plasmid to the microcentrifuge tube
7. Chill in ice for 20 minutes, then heat in the heat bath for 35 seconds, then chill again for 2 minutes
8. Sterilize the area and instruments with ethanol/water mixture, and prepare a flame
9. Add 200μL of LB broth into microcentrifuge tube
10. Shake in the 37°C shaker for 1 hour
11. Place the plate with the corresponding antibiotic in the 37°C incubator around the same time you place the microcentrifuge tube in the shaker
12. Sterilize the area and instruments with ethanol/water mixture, and prepare a flame
13. Pipette 100µL of the transformed cells onto the plate
14. Spread around with beads, making sure to not have the beads circle the outside
15. Dump beads in bleach and place plate in the 37°C incubator overnight
16. Return the GenHog cells

Materials
1. Transformed colonie(s)
2. Corresponding antibiotic(s)
3. LB media
4. Wooden inoculation stick
5. Plastic culture tube
6. 37 °C shaker
Procedure
1. Obtain LB broth, the correct antibiotic, and the plate with the desired colonies
2. Label plastic culture tube, create flame, and sterilize pipette, hands, tips, and bench
3. Pipette 5mL LB into the culture tube
4. Add 5µL the antibiotic to the LB (1:1000 ratio of antibacterial to LB)
5. Sterilize wooden stick
6. Grab a colony by touching the wooden stick to a lonely dot.
7. Place the stick (colony-end) into the LB solution and shake well
8. Shake at 250rpm in 37°C overnight
9. Return the LB broth, antibiotic, and plate

Materials
1. P1 buffer
2. P2 buffer
3. P3 buffer
4. Microcentrifuge Tubes
5. Filter Columns & Tubes
6. Endo-Wash Buffer
7. Zippy-Wash Buffer
8. Autoclaved Nanowater
Procedure
1. Ensure that the cultures are in a plastic culture tube. Place tubes in the centrifuge, balance the centrifuge, and run for 10 minutes at 5000 g
2. After centrifuging, dump out LB broth into broth waste and use a pipette to remove any remaining broth. Make sure not to touch the cells
3. Pipette 200 µL of P1 buffer (Pink) to the culture tubes
4. After adding P1 buffer, pipette the solution up and down repeatedly to break up the pellet
  1. Tilt the tube at an angle so the buffer isn’t covering the pellet. Use the pipette to extract the buffer and release it over the pellet
5. Transfer the buffer and pellet solution to appropriately labeled microcentrifuge tubes
6. Pipette 200 µL of P2 buffer (Blue) into the microcentrifuge tubes
  1. Invert microcentrifuge tubes twice
  2. Let sit for 1-2 minutes
7. Pipette 400 µL of P3 buffer (Yellow) into the microcentrifuge tubes
  1. Obtain P3 buffer from the fridge and replace immediately after using
  2. Invert tubes repeatedly until the solution turns entirely yellow
8. Centrifuge tubes for 5 minutes at max speed
  1. While centrifuging, set up filter column found in miniprep kit
9. Transfer supernatant from microcentrifuge tubes into filter tubes
  1. Use a pipette to remove all of the supernatant, taking care not to touch any of the solid left in the microcentrifuge tubes
10. Centrifuge filter tubes for 30 seconds at 16,000 rpm
  1. Discard the flowthrough in the biohazard waste
11. Add 200 µL of the Endo-Wash buffer to the filter tubes and centrifuge at 16,000 rpm for 30 seconds
  1. Discard the flowthrough
12. Add 400 µL of the Zippy-Wash buffer to the filter tubes and centrifuge at 16,000 rpm for 30 seconds
13. Centrifuge for an additional 2 minutes at 20,000 g
  1. Discard flowthrough
14. Label new microcentrifuge tubes with the plasmid name
  1. Plasmid: pSB3K3 & pSB4A5
15. Place filter tubes into new microcentrifuge tubes
16. Add 30 µL of autoclaved Nanowater to the filter tubes
  1. Let stand for 1 minute
17. Centrifuge filter tubes for 1.5 minutes at 16,000 g
18. Find DNA concentration(X) using NanoDrop

Materials
1. PCR Tubes
2. EcoR1
3. Pst1
4. Cutsmart Buffer
Procedure
1. Obtain and label PCR tubes
2. Add the following components to the tube:
  1. 1 µL plasmid
    1. Calculate the volume of plasmid required for 1 µg
  2. 1 µL of EcoR1
  3. 1 µL of Pst1
  4. 5 µL of Cutsmart buffer
  5. X µL of autoclaved Nanowater
    1. X = water required to bring the total volume to 50 µL
3. Set tube in thermal cycler
  1. Run using “Digest” setting---37 degrees Celsius, 3 hours
Calculations
1. 1000 ng * ^{1 L}⁄_{x ng} = µL plasmid
  X = concentration of plasmid

Materials
1. 1x TAE Buffer
2. Agarose
3. Ethidium Bromide
4. Gel Holder/ Well comb
5. Electrophoresis Machine
6. DNA ladder
7. DNA dye
8. Imaging Apparatus/Software
Procedure
1. Add 50 mL of 1x TAE and 0.75 g of agarose to the correct flask
  1. Place a smaller flask upside down inside the larger flask, covering it
2. Place flask in microwave for 1.5 minutes at power level 10
3. Allow agarose to cool for 10 minutes
4. Add 4 µL of ethidium bromide inside the fume hood
  1. Ethidium bromide is a carcinogen; do not touch or inhale
  2. Dispose of all tips in the ethidium bromide tip container
5. Pour agarose into a gel holder and place the appropriately-sized well comb
  1. Pop any bubbles in the agarose
  2. Allow to cool for 20 minutes
  3. Insert the gel tray into the gel box with the wells close to the black electrode
6. Use arrow alongside gel box to orient tray if needed; DNA will move from the black electrode to the red electrode
  1. “Run to red”
  2. Pour enough 1x TAE to cover the gel and reach the max fill line
  3. Choose your DNA ladder based on how large you expect the DNA fragments to be
  4. Obtain DNA ladder from fridge
  5. Obtain gel loading dye from tray near electrophoresis equipment
7. Note: both the DNA ladder and the loading dye are purple. Do not confuse them!
  1. Add loading dye to the DNA sample
8. Add dye to DNA in a 1:6 dilution
  1. For example, add 10 µL of dye to 50 µL of DNA
  2. Pipette the DNA/dye solution up and down to mix
  3. Add 8 µL of the ladder to well 1
  4. Place up to 30 µL of DNA into wells 2-8 as needed
  5. Place lid on gel box and connect to a volt machine.
9. Set to 100 volts for 60 minutes and hit run
  1. Bubbles should appear if the electrophoresis is working
10. Time depends on the size of DNA fragments. Check gel periodically to make sure the DNA does not run off the end of the gel
  1. Place gel in ChemiDoc
  2. Open ImageLab3 Software
  3. Select ethidium bromide
  4. Adjust filter based on instructions from the software
  5. Use Image Tools to crop the picture
  6. Adjust contrast to better see the gel if desired
  7. Use Annotation Tools to create notes
  8. Ladder type
  9. Digestion product
  10. Digestion enzyme names
  11. Analyze gel to determine if the gene were successfully excised from the plasmid

Materials
1. Gel Recovery Kit
2. Centrifuge
3. Microcentrifuge Tubes
4. Autoclaved Nanowater
5. Nanodrop
Procedure
1. Use UV lamp with face shields to visualize bands in gel
2. Use razor to carefully cut the bands out of the gel
  1. Store them in their own microcentrifuge tubes (labeled)
3. Dispose of excess gel and gloves in special container
  1. This is due to the ethidium bromide
4. Determine and record the weight of the recovered gels
  1. Use an empty microcentrifuge tube to tare the balance
5. Retrieve the DNA Recovery Kit
6. Add X amount of Agarose dissolving buffer(ADB) to the respective microcentrifuge tube
  1. X = 3000 * (gel weight)
    1. EX: 3000 * (0.126 g) = 378 µL
  2. The max amount of ADB that can be added is 750 µL. If the gel weighs more than 0.250 g, then more gel must be cut off or the gel must be divided into two separate tubes
7. Place the microcentrifuge tubes in a 50-55 °C water bath for 10 minutes
  1. Make sure the gel has completely dissolved, if there are pieces present, put back in the water bath for a couple minutes
8. Vortex to mix well
9. Retrieve and label collection tubes and their filter counterparts
10. Transfer the liquid into the filter using a pipette
11. Centrifuge for 30 s at 16,000 G
  1. Discard flowthrough
12. Add 200 µL of wash buffer into the filter and centrifuge again with the same conditions
13. Repeat the wash buffer step
14. Centrifuge again for a minute at 21,000 G
15. Move the filters into new, labeled microcentrifuge tubes
16. Add 10 µL of autoclaved nanowater and centrifuge for 1 min at 16,000 G
17. Check and record the concentration with the nanodrop machine

Materials
1. BBa_K584000 cells
2. BBa_K206000 cells
3. L-arabinose
4. Autoclaved NanoWater
5. LB Media
6. 1x PBS
7. Centrifuge
8. Plastic culture tubes
9. Plate reader
10. Flow cytometer
Procedure
1. Remove inoculated cultures from shaker and test the OD₆₀₀ using 100 µL and the plate reader.
2. Perform a dilution for each sample into 10 individual tubes (triplicates of 3 conditions per sample and 1 tube to monitor OD growth) with more LB to OD₆₀₀ 0.05 and let culture until 0.1. The new volume should be 1 mL. There should be 20 tubes total. Antibiotics should be added also.
  1. (OD1)(x)=(0.05)(1mL)
    x = volume from culture
3. After OD₆₀₀ is 0.1, use 500 µL from each of the two cultures to find the starting OD₅₉₅ and fluorescence with the plate reader
  1. Use a clear plate to measure OD₅₉₅and a black plate to measure fluorescence
  2. Excitation & emission - http://parts.igem.org/wiki/index.php?title=Part:BBa_E004
    1. 490 nm excitation
    2. 525 nm emission
4. Wash and resuspend cells in PBS for initial measurement
  1. Cool centrifuge to 4 °C, use plastic culture tubes, and centrifuge at 5,000 g for 10 minutes
  2. Discard supernatant
  3. Add 500 µL of PBS and resuspend
5. Add 5 mL of the correct culture into each of the 6 culture tubes (BBa_K584000 should be in 3 of the tubes, BBa_K206000 in the other 3) Add culture, 20% L-arabinose, and/or autoclaved Nanowater in the following proportions:
  1. 0%
    1. 5 mL BBa_K584000/BBa_K206000
    2. 555 µL autoclaved Nanowater
  2. 0.2%
    1. 5 ml BBa_K584000/BBa_K206000
    2. 55.5 µL 20% L-arabinose
    3. 499.5 µL autoclaved Nanowater
  3. 2%
    1. 5 mL BBa_K584000/BBa_K206000
    2. 555 µL 20% L-arabinose
6. Allow cultures to grow for 1 hour in the shaker at 250 rpm and 37 °C
7. Remove 500 µL from each culture after 1 hour and wash and resuspend with PBS
  1. Cool centrifuge to 4 °C, use plastic culture tubes, and centrifuge at 5,000 g for 10 minutes
8. Discard supernatant
9. Add 500 µL of PBS and resuspend
10. Use 100 µL from each of the cultures to find the OD₅₉₅ and fluorescence with the plate reader
11. Save 300 µL from each culture for flow cytometry
  1. Place 300 µL sample in 4 °C refrigerator to halt growth
12. Repeat steps 6-9 four more times to generate time points every hour for five hours
13. Place cultures on ice and transport to flow cytometry lab on UNL East Campus
Calculations
1. BBa_K206000 Culture Dilution
  1. (1.8 OD) (x mL) = (0.05 OD) (20 mL)
    x = 0.556 mL = 556 L original culture
2. BBa_K584000 Culture Dilution
  1. (1.5 OD) (x mL) = (0.05 OD) (20 mL)
    x = 0.667 mL = 667 L original culture

Materials
1. Cells
2. Premade glycerol tubes
Procedure
1. Obtain glycerol tubes and label appropriately
  1. pBAD + CheZ #1 7/24/19
  2. pBAD + CheZ + YbaQ #1 7/24/19
  3. pBAD + GarKS #1 7/24/19
  4. pBAD + GarKS + NSP4 #1 7/24/19
  5. P2 + GarKS + NSP4 #1 7/24/19
2. Add 100 µL of inoculated culture to 100 µL of glycerol
3. Store glycerol tubes in -80 °C freezer for future use

Materials
1. LB Broth
2. Nanowater
3. Autoclave
Procedure
1. Weigh out 25g/1L of LB broth
2. Add nanowater to complete the liter
3. Autoclave under lab solutions setting for one hour

Materials
1. Yeast Extract
2. Tryptone
3. Glycerol
4. LB broth powder
5. Agar powder
6. 10X PBS
7. Nanowater
Procedure
1. Add the following quantities together in a 250 mL bottle for TB:
  1. Yeast Extract: 6 g
  2. Tryptone: 5 g
  3. Glycerol: 1 mL
  4. Nanowater: 225 mL
  5. 10X PBS should be mixed in before use: 25 mL
2. Add the following quantities together in a 250 mL bottle for LB broth:
  1. LB broth powder: 6.25 g
  2. Nanowater: 250 mL
3. Add the following quantities together in a 500 mL bottle for LB agar:
  1. LB broth powder: 12.5 g
  2. Agar powder: 7.5 g
  3. Nanowater: 500 mL
4. Mix all the contents well
5. Place in autoclave with proper precautions on the lab solution setting for one hour

Materials
1. PCR tubes
2. dNTPs
3. 10x buffer
4. MgSO4
5. KOD Polymerase
6. Autoclaved Nanowater
7. Primers
8. DNA parts
Procedure
1. Create the primer mixtures by combining the following components:
  1. 1 μL forward primer
  2. 1 μL reverse primer
  3. 8 μL autoclaved NanoWater
2. Create DNA templates by combining the following components:
  1. 1 μL stock pBAD+GarKS+NSP4 template (100 ng/μL)
  2. 9 μL autoclaved NanoWater
3. Create a 50 μL PCR mixture by adding the following components to a PCR tube:
  1. 32.5 μL autoclaved Nanowater
  2. 5 μL dNTPs
  3. 5 μL 10x Buffer
  4. 3 μL MgSO₄
  5. 2 μL DNA template
  6. 1.5 μL Primer Mix
  7. 1 μL KOD Polymerase
    1. Be sure to add the KOD polymerase last; the order of the other components can be changed
4. Open the thermal cycler and select the KOD protocol
5. Modify the time for the 70 °C cycle to match the length of the template
  1. Set time to 18 seconds for the two garvicin parts
6. Plate both PCR tubes in thermo cycler, close the lid, and press start

Materials
1. Autoclaved nanowater
2. NEBuilder HiFi DNA Assembly Master Mix
3. PCR tubes
4. Chemically-competent cells
5. SOC media
6. Chloramphenicol plates
7. Culture beads
8. 37 °C incubator
9. 37 °C shaker
10. Hot water bath
11. Ice bath
12. Thermocycler
Procedure
1. Obtain parts, vector, nanowater, master mix, and PCR tubes
  1. Thaw and vortex master mix thoroughly and keep on ice
2. Calculate amounts needed for a vector:insert ratio of 1:1 and a total fragment amount of 0.2 - 0.5 pmols (found in calculations section)
3. Dilute each part with 10 µL of autoclaved nanowater to make an approximate concentration of 100 ng/µL
4. Add the following quantities to the PCR tube (total of 20 µL):
  1. Insert: x
  2. Vector: x
  3. Autoclaved nanowater: x
  4. HiFi DNA Assembly Master Mix: 10 µL
5. Incubate samples in a thermocycler at 50°C for 60 minutes. Once completed, store on ice or at -20°C
6. Once completed, use right away or store at -20°C. Thaw chemically-competent cells on ice when ready to use the samples.
7. Add 2 µL of the assembled product to the competent cells. Mix gently by pipetting up and down or by flicking the tube 4-5 times. Do not vortex.
8. Place the mixture on ice for 30 minutes. Do not mix.
9. Heat shock at 42°C for 30 seconds. Do not mix.
10. Transfer tubes to ice for 2 minutes
11. Add 950 µL of room-temperature SOC media to the tube
12. Incubate the tube at 37°C for 60 minutes. Shake vigorously (250 rpm)
13. Warm chloramphenicol plates to 37°C
14. Add 100 µL of cells to the plate, and use beads to spread out the cells
15. Incubate overnight at 37°C
Calculations
1. Example:
  Improved frag1=1.01pmoles→Dilute w/10 µL→~100ng/µL and 0.10pmols
  Improved frag2=1.40pmoles→Dilute w/10 µL→~100ng/µL and 0.14pmols
  Improved frag3=1.64pmoles→Dilute w/10 µL→~100ng/µL and 0.16pmols
  pSB1C3 = 25ng/µL → 4µL = 100ng & 0.07 pmol
  Total mix = 7µL, 1:1 ratio in ng, 0.47 pmol < 0.50 pmol

Materials
1. DNA sample
2. Sequencing tube
3. 2 µM Forward primers
  1. (100 M) (x) = (2 M) (50 L)
    x = 1 L primer stock
Procedure
1. Combine the following components into each tube: 12μL total
  1. DNA: (280ng)(1μL/xng)
  2. 4 µL of 2 µM Forward Primer
  3. Autoclaved Nanowater(8µL - volume of DNA)
2. Send tube to Eurofin for sequencing

Materials
1. Autoclaved Nanowater H20
2. Tryptone
3. NaCl
4. Agarose
Procedure
1. Combine the following components in 500 mL of autoclaved nanowater H20:
  1. 5.00 g tryptone
  2. 2.5 g NaCl
  3. 1.25 g agarose
2. Autoclave media for 20 minutes at 120°C

Materials
1. Qiagen Resuspension Buffer (Buffer P1)
2. Qiagen Lysis Buffer (Buffer P2)
3. Qiagen Neutralization Buffer (Buffer P3)
4. Qiagen Neutralization Buffer II (Buffer N3)
5. Qiagen Binding Buffer (Buffer PB)
6. Qiagen Wash Buffer (Buffer PE)
7. Qiagen Elution Buffer (Buffer EB)
Procedure
1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. For centrifuge processing, follow the instructions marked with a triangle. For vacuum manifold processing, follow the instructions marked with a circle. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source.
  1. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source.
  2. Note: This step is only required when using endA+ strains or other bacteria strains with high nuclease activity or carbohydrate content.
7. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through, or apply vacuum to the manifold to draw the solution through the QIAprep 2.0 spin column and switch off the vacuum source. Transfer the QIAprep 2.0 spin column to the collection tube.
8. Centrifuge for 1 min to remove residual wash buffer.
9. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM TrisCl, pH 8.5) or water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.
10. If the extracted DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

Materials
1. CaCl₂ 2H2O (F.W. 147.01) - 1.18 g per 100mL solution
2. MnCl₂ 4H₂O (F.W. 197.91) - 0.4 g per 100mL solution
3. MgCl₂ 6H₂O (F.W. 203.30) - 0.2 g per 100mL solution
4. Potassium acetate (F.W. 98.14) - 1mL of a 1 M(pH 7.0) per 100mL solution
5. Redistilled glycerol- 10 mL per 100mL solution
Procedures
1. Note: It is absolutely critical to maintain sterile conditions throughout the procedure.
2. Mix the materials together.
  1. Dilute to 100mL solution with Autoclaved nanowater
3. Adjust the final solution to pH 6.4 with o.1 N HCl.
4. Filter the CCMB solution through 0.2 μm filter and kept at 4°C.

Materials
1. CCMB Solution
2. Colony of strain of interest
Procedures
1. Day One
  1. Inoculate 5mL of LB with a single colony of the strain of interest.
  2. Grow the culture overnight at 37 * C with shaking.
2. Day Two
  1. Adjust the temperature of a floor shaker to 30°C. It is absolutely critical to maintain the temperature no higher than 30°C. Prior to proceeding to the next step, the chamber temperature should be stabilized for no less than 30 min.
  2. Inoculate 100 mL of LB containing appropriate antibiotic with 1.0 mL of the overnight culture.
  3. Grow the cells at < 30°C with shaking until the OD₆₀₀ of the culture reaches 0.4-0.6. Depending on the strain, this step normally takes more than two hours.
  4. While the cells are growing, chill the CCMB stock solution on ice. Also turn on the centrifuge and chill the rotor to 4°C. Label 40 of 1.5 mL sterile microtubes with the name of the strain and chilled them in the freezer. It is absolutely critical to have cold instruments and solutions for successfully carrying out the procedure.
  5. When the OD₆₀₀ of the cell culture reaches the desired value, collect the cells by centrifugation at 5,000 g at 4°C for 10 min for two times in a 50 mL sterile centrifuge tubes.
  6. It’s absolutely critical to maintain the cell pellet cold for the rest of the procedure. Quickly pour away the supernatant and try to remove as much the residual medium as possible using pipet.
  7. Keep the cell pellet on ice, add 1 mL of chilled CCMB solution and resuspend the cells. Collect the cells by centrifugation at 5,000 g at 4°C for 10 min.
  8. Quickly remove all the supernatant using a pipet.
  9. Completely resuspend the cell pellets in 4 mL of cold CCMB solution.
  10. Transfer aliquots (0.1 mL) of the competent cells to sterile, labeled, chilled 1.5 mL microtubes. Leave the competent cells on ice for half an hour. Then transfer the tubes into a storage box and store the cells at -80°C. The transformation efficiency is boosted after one cycle of freeze. Check the transformation efficiency of the freshly made competent cells (This method normally yields transformation efficiency OD₆₀₀ cfu/μg DNA). Cells prepared using this method maintain competency for at least 6 months.
  11. Return the CCMB solution back to the fridge for storage.

Materials
1. E. coli strain UU2685 (ΔcheZ)
2. pBAD + CheZ DNA construct
3. pBAD + CheZ + YbaQ DNA construct
4. 85 mm Petri dishes
5. Tryptone
6. Agarose
7. L-arabinose
8. 37 °C shaker
9. 30 °C incubator
10. Streptomycin (50 µg/mL)
Procedures
1. Prepare diffusion plates
  1. Mark empty plates with a 15 mm line
  2. Place a small dot in the middle of the 15 mm line
  3. Create approximately 600 mL of tryptone agar for plates
    1. All agar should contain 1% tryptone, 0.25% agarose, and 50 µg/mL streptomycin
    2. Add 0.02% arabinose to a 120 mL portion of agar
    3. Add 0.2% arabinose to 180 mL portion of agar
  4. Pour the following plates:
    1. 6 plates 0% arabinose
    2. 6 plates 0.02% arabinose
    3. 9 plates 0.2% arabinose
2. Prepare directionality plates
  1. Mark empty plates with a 15 mm line
  2. Place a small dot in the middle of the 15 mm line
  3. Draw two 15 mm boxes on each end of the 15 mm line
    1. Label one box “Tryptone” and the other box “Arabinose”
  4. Create tryptone agar for plates
    1. Agar should contain 1% tryptone, 0.25% agarose, and 50 µg/mL streptomycin
  5. Pour 21 plates
3. Prepare cells
  1. Transform the following DNA constructs into E. coli strain UU2685 (ΔcheZ):
    1. pBAD + CheZ
    2. pBAD + CheZ + YbaQ
  2. After transformation, prepare three inoculations each for the following cell types using LB media:
    1. UU2685 from stock (no transformation)
    2. pBAD + CheZ from transformed colonies
    3. pBAD + CheZ + YbaQ from transformed colonies
  3. Place inoculation in a 37 °C shaker at 250 rpm overnight
  4. The following day, measure the OD of the inoculations and dilute with LB to an OD of 0.05
  5. Allow the cultures to grow to an OD of 0.5
  6. Remove cultures from shaker; plate cells for diffusion and directionality assays
4. Diffusion assay
  1. Plate 5 µL of UU2685 onto a 0% arabinose plate. Place the cells on the dot labeled in the middle of the plate.
  2. Allow the cells to dry
  3. Place the plates in a 30 °C incubator overnight
  4. Repeat steps 1-3 for all cell cultures to create the following plates:
    1. UU2685
      1. Three 0% arabinose plates
      2. Three 0.02% arabinose plates
      3. Three 0.2% arabinose plates
    2. pBAD + CheZ
      1. Three 0% arabinose plates
      2. Three 0.02% arabinose plates
      3. Three 0.2% arabinose plates
    3. pBAD + CheZ + YbaQ
      1. Three 0% arabinose plates
      2. Three 0.02% arabinose plates
      3. Three 0.2% arabinose plates
5. Directionality
  1. Plate 5 µL of UU2685 onto the dot in the center of the plate
  2. In the box labeled “Tryptone,” place three 2 µL aliquots of tryptone in equally spaced increments
  3. In the box labeled “Arabinose,” place three 2 µL aliquots of water in equally spaced increments. Use water as the 0% arabinose condition
  4. Allow the cells, tryptone, and water to dry.
  5. Place the plates in a 30 °C incubator overnight
  6. Repeat steps 1-5 for all cell cultures to create the following plates:
    1. UU2685
      1. Three 0% arabinose plates
      2. Three 0.02% arabinose plates
      3. Three 0.2% arabinose plates
    2. pBAD + CheZ
      1. Three 0% arabinose plates
      2. Three 0.02% arabinose plates
      3. Three 0.2% arabinose plates
    3. pBAD + CheZ + YbaQ
      1. Three 0% arabinose plates
      2. Three 0.02% arabinose plates
      3. Three 0.2% arabinose plates
6. The next day
  1. Use the 15 mm lines marked on the plates to determine the radius of growth

Materials
1. BBa_K1022100 cells
2. BBa_K3191101 cells
3. BBa_K3191102 cells
4. BBa_K206000 cells
5. L-arabinose
6. AIP
7. Autoclaved NanoWater
8. LB Media
9. 1x PBS
10. Centrifuge
11. Plastic culture tubes
12. Plate reader
13. Flow cytometer
Procedure
1. Remove inoculated cultures from shaker and test the OD₆₀₀ using 100 µL and the plate reader.
2. Perform a dilution for each sample into 13 individual tubes (triplicates of 4 conditions per sample and 1 tube to monitor OD growth) with more LB to OD₆₀₀ 0.05 and let culture until 0.1. The new volume should be 1 mL. There should be 52 tubes total. Antibiotic should be added also.
  1. (OD1)(x)=(0.05)(1mL)
    x = volume from culture
3. After OD₆₀₀ is 0.1, add the correct amounts of AIP and 20% L-arabinose for each condition:
  1. AIP & arabinose:
    1. AIP: 4 µL
    2. 20% arabinose: 5 µL
  2. AIP:
    1. AIP: 4 µL
    2. 20% arabinose: 0 µL
  3. arabinose:
    1. AIP: 0 µL
    2. 20% arabinose: 5 µL
  4. Neither:
    1. AIP: 0 µL
    2. 20% arabinose: 0 µL
4. Remove 300 µL from each of the cultures; these is the hour 0 samples
5. Place the cultures in the shaker at 250 rpm and 37 °C for 8 hours
6. Wash and resuspend the hour 0 cells in PBS for initial measurement
  1. Centrifuge at 5,000 g for 10 minutes
    1. Cool centrifuge to 4 °C
    2. Use plastic culture tubes
  2. Discard supernatant
  3. Add 300 µL of PBS and resuspend
7. Put 100 µL from each of the cultures into a clear 96 well plate and a black 96 well plate to find the OD₆₀₀ and fluorescence with the plate reader
8. Remove 300 µL from each culture after 8 hours and wash and resuspend with PBS
  1. Centrifuge at 5000 g for 10 minutes
    1. Cool centrifuge to 4 °C
    2. Use plastic culture tubes
  2. Discard supernatant
  3. Add 300 µL of PBS and resuspend
9. Put 100 µL from each of the cultures into a clear 96 well plate and a black 96 well plate to find the OD₆₀₀ and fluorescence with the plate reader
10. Save the rest of the cultures for flow cytometry
  1. Place samples in 4 °C refrigerator to halt growth
11. Place cultures on ice and transport to flow cytometry lab
Calculations
1. AIP:
  1. (2500 µM)(v) = (10 µM)(1000 µL) v = 4 µL
2. Arabinose:
  1. (20%)(v) = (0.2%)(1000 µL) v = 10 µL

Materials
1. 1 L M9 broth
2. Three 1 L Erlenmeyer flasks
3. Rotovap
4. 37 °C shaker
5. Dry ice
6. Acetone
7. Ethyl acetate
8. 25 or 50 mL round bottom flask
9. 20% L-arabinose sterile solution
10. Kanamycin (50 µg/mL)
11. 50 mL sterile conical tubes
12. Autoclaved nanowater
13. 50% acetonitrile, 5% formic acid, 45% H2O solution
14. Inoculating loops
15. Plastic culture tubes
Procedures
1. Prepare culture tubes with 5 mL M9 broth, 5 μL kanamycin
2. Inoculate GarKS strains and pSB3K3 negative control from glycerol stock into culture tubes
3. Grow to an OD equal to or greater than 2.0
  1. Check OD with a spectrophotometer using a 1 mL M9 broth as the blank and a 1:10 dilution of culture. Dilute using M9 broth.
4. Place the entire culture into a 1 L flask containing 250 mL of M9 broth
5. Incubate the large flasks in a 37 °C shaker at 250 rpm until OD reaches 0.5
6. Induce with L-arabinose to create a 0.2% concentration in culture medium
7. Allow cells to grow to OD of 2.0 following induction of L-arabinose
8. Pour the cell culture into a 250 mL centrifuge bottle
9. Harvest cells by centrifugation at 5500 rpm for 10 minutes at 4 °C
10. Pour the supernatant into new 250 mL centrifuge bottle
11. Use 5 mL of supernatant to resuspend the cell pellet in the original bottle
12. Transfer pellet resuspension to a 50 mL conical tube
13. Centrifuge the 50 mL conical tube at 5500 rpm for 10 minutes at 4 °C
14. Pour the supernatant back into the 250 mL centrifuge bottle in step 10
15. Store the cell pellet at -80 °C and store the supernatant at 4 °C for later use
16. Pour the supernatant into a 1 L round bottom flask
17. Rotovap the supernatant to decrease the volume to approximately 7 mL
18. Pour the supernatant into a new 50 mL conical tube
19. Rinse the round bottom flask with three 1 mL portions of autoclaved nanowater and add the rinse to the 50 mL conical tube. This creates a total volume of 10 mL
20. Store the conical tube at -80 °C for future analysis
21. Thaw the supernatant in the 50 mL conical tube and aliquot 3 mL into a 25 mL or 50 mL round bottom flask
22. Rotovap to dryness
23. Create 10 mL of the 50% acetonitrile, 5% formic acid, 45% H20 solution
24. Add 500 µL of acetonitrile/formic acid solution to dissolve the protein
25. Incubate at room temperature for 1 hour; store at 4 °C for Bradford assay or other analysis

Materials
1. Bio-Rad protein assay
2. 96 well plate
3. Plate reader
Procedure
1. Prepare the following standard solutions with bovine serum albumin (BSA) and autoclaved nanowater:
  1. 0 mg/mL BSA
  2. 0.125 mg/mL BSA
  3. 0.25 mg/mL BSA
  4. 0.50 mg/mL BSA
  5. 0.75 mg/mL BSA
  6. 1.0 mg/mL BSA
2. Dilute the protein sample with autoclaved nanowater to create the following dilutions:
  1. 1:30 dilution
  2. 1:20 dilution
  3. 1:10 dilution
  4. 5:5 dilution
3. Prepare a sample for the assay by adding 5 µL of the desired sample to 250 µL of Coomassie Blue dye. Pipet to mix.
  1. Create three samples for each standard solution and 3 samples for each protein dilution to allow triplicate measurements
4. Place the samples in a 96 well plate and incubate at room temperature for 10 minutes. Keep the sample in the dark during the incubation period.
5. After 10 minutes, place the 96 well plate in a plate reader to measure the OD at 595 nm
6. Use the absorbance values for the BSA standards to generate a standard curve of absorbance vs. concentration
7. Use the equation of the standard curve and the absorbance value of the protein sample to find the concentration of protein in a given dilution
8. Use the dilution values to calculate the concentration of protein in the undiluted sample

Materials
1. L-arabinose powder
2. Nanowater
3. Sterilized syringe
4. Sterile filter for syringe
5. 50 mL conical tube
Procedure
1. Sterilize workbench and ignite flame
2. Disolve 10 g of L-arabinose powder in 50 mL of nanowater
3. Load a sterilized syringe with appropriate sterile filter and remove syringe plug
4. Load the solution into the syringe.
5. Once filled, take syringe plug and push the solution through the filter into a couple 50 mL conical tubes

Materials
1. Na₂HPO₄-7H₂O
2. KH₂PO₄
3. NaCl
4. NH₄Cl
5. Autoclaved Nanowater
Procedure
1. Add the following quantities into a 1 L bottle:
  1. 32g Na₂HPO₄-7H₂O
  2. 7.5g KH₂PO₄
  3. 1.25g NaCl
  4. 2.5g NH₄Cl
  5. 500 mL nanowater
2. Autoclave the solution

Materials
1. 1M MgSO4 solution
2. 1M CaCl2 solution
3. glycerol
4. M9 salt solution
5. 0.3M Leucine (in 1M HCl solution) (filter sterilized)
Procedure
1. Autoclave the MgSO4, CaCl2, and M9 salt solutions
2. Add the following quantities together:
  1. 2 mL of autoclaved 1M MgSO4 solution
  2. 100µL of autoclaved 1M CaCl2 solution
  3. 20 mL of glycerol
  4. 200 mL of autoclaved M9 salt solution
  5. 0.5 mL of 0.3M Leucine (in 1M HCl solution) (filter sterilized)

Materials
1. T4 buffer
2. T4 ligase
3. Insert
4. Vector
5. Autoclaved Nanowater
Procedure
1. Look for the insert size removed from the plasmid and subtract it from the plasmid size to determine the new plasmid size after digestion
  1. Insert construct size: _____________bp
  2. Plasmid size: ____________ bp – insert size=______________ bp
2. Determine the insert 3:1 ratio using the following formula:
  1. ^{insert size bp}⁄_{digested plasmid bp} * 3 = insert bp 3 : 1 ratio * 1 μL= x μL
3. Using the results from the mentioned formula determine the nanograms needed for a concentration of 50 ng µL^-1
  1. ^{x μL}⁄₁ * ^{50 ng}⁄_1μL = x ng
4. Determine the volume needed from the construct (insert) using the following formula:
  1. ^{x μL}⁄₁ * ^{1 μL}⁄_{concentration of ng µL^-1} = x μL of construct needed
5. determine the volume needed from the plasmid (vector) using the following formula:
  1. ^{50 μL}⁄₁ * ^{1 μL}⁄_{plasmid concentration ( ng µL^-1)} = x μL of plasmid needed
6. Prepare the following mix for reaction for 10μL
  1. T4 buffer 1μL
  2. T4 ligase 0.5μL
  3. Insert x
  4. Vector x
  5. Autoclaved Nanowater x

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