iGEM 2019 — Team UNebraska-Lincoln

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# Team:UNebraska-Lincoln/Notebook

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# Notebook


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# Team:UNebraska-Lincoln/Experiments

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# Experiments

Making Plates

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

Transformation (GeneHogs)

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

Inoculation

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

Miniprep (Zyppy Plasmid Miniprep Kit)

1. Materials
   1. P1 buffer- P2 buffer- P3 buffer- Microcentrifuge Tubes- Filter Columns & Tubes- Endo-Wash Buffer- Zippy-Wash Buffer- 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- 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- Pipette 200 µL of P1 buffer (Pink) to the culture tubes- 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- Transfer the buffer and pellet solution to appropriately labeled microcentrifuge tubes- Pipette 200 µL of P2 buffer (Blue) into the microcentrifuge tubes
                  1. Invert microcentrifuge tubes twice- Let sit for 1-2 minutes- Pipette 400 µL of P3 buffer (Yellow) into the microcentrifuge tubes
                    1. Obtain P3 buffer from the fridge and replace immediately after using- Invert tubes repeatedly until the solution turns entirely yellow- Centrifuge tubes for 5 minutes at max speed
                      1. While centrifuging, set up filter column found in miniprep kit- 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- Centrifuge filter tubes for 30 seconds at 16,000 rpm
                          1. Discard the flowthrough in the biohazard waste- 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- Add 400 µL of the Zippy-Wash buffer to the filter tubes and centrifuge at 16,000 rpm for 30 seconds- Centrifuge for an additional 2 minutes at 20,000 g
                                1. Discard flowthrough- Label new microcentrifuge tubes with the plasmid name
                                  1. Plasmid: pSB3K3 & pSB4A5- Place filter tubes into new microcentrifuge tubes- Add 30 µL of autoclaved Nanowater to the filter tubes
                                      1. Let stand for 1 minute- Centrifuge filter tubes for 1.5 minutes at 16,000 g- Find DNA concentration(X) using NanoDrop

Digestion

1. Materials
   1. PCR Tubes- EcoR1- Pst1- Cutsmart Buffer- Procedure
     1. Obtain and label PCR tubes- Add the following components to the tube:
          1. 1 µL plasmid
             1. Calculate the volume of plasmid required for 1 µg- 1 µL of EcoR1- 1 µL of Pst1- 5 µL of Cutsmart buffer- X µL of autoclaved Nanowater
                     1. X = water required to bring the total volume to 50 µL- 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

Gel Imaging

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

Gel Recovery

1. Materials
   1. Gel Recovery Kit- Centrifuge- Microcentrifuge Tubes- Autoclaved Nanowater- Nanodrop- Procedure
     1. Use UV lamp with face shields to visualize bands in gel- Use razor to carefully cut the bands out of the gel
          1. Store them in their own microcentrifuge tubes (labeled)- Dispose of excess gel and gloves in special container
            1. This is due to the ethidium bromide- Determine and record the weight of the recovered gels
              1. Use an empty microcentrifuge tube to tare the balance- Retrieve the DNA Recovery Kit- 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- 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- 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- Vortex to mix well- Retrieve and label collection tubes and their filter counterparts- Transfer the liquid into the filter using a pipette- Centrifuge for 30 s at 16,000 G
                            1. Discard flowthrough- Add 200 µL of wash buffer into the filter and centrifuge again with the same conditions- Repeat the wash buffer step- Centrifuge again for a minute at 21,000 G- Move the filters into new, labeled microcentrifuge tubes- Add 10 µL of autoclaved nanowater and centrifuge for 1 min at 16,000 G- Check and record the concentration with the nanodrop machine

Fluorescence Characterizations

1. Materials
   1. BBa\_K584000 cells- BBa\_K206000 cells- L-arabinose- Autoclaved NanoWater- LB Media- 1x PBS- Centrifuge- Plastic culture tubes- Plate reader- Flow cytometer- Procedure
     1. Remove inoculated cultures from shaker and test the OD600 using 100 µL and the plate reader.- 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- 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 OD595 and a black plate to measure fluorescence- Excitation & emission - [http://parts.igem.org/wiki/index.php?title=Part:BBa\_E004](http://parts.igem.org/wiki/index.php?title=Part:BBa_E0040)
                 1. 490 nm excitation- 525 nm emission- 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- Discard supernatant- Add 500 µL of PBS and resuspend- 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- 555 µL autoclaved Nanowater- 0.2%
                     1. 5 ml BBa\_K584000/BBa\_K206000- 55.5 µL 20% L-arabinose- 499.5 µL autoclaved Nanowater- 2%
                       1. 5 mL BBa\_K584000/BBa\_K206000- 555 µL 20% L-arabinose- Allow cultures to grow for 1 hour in the shaker at 250 rpm and 37 °C- 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- Discard supernatant- Add 500 µL of PBS and resuspend- Use 100 µL from each of the cultures to find the OD595 and fluorescence with the plate reader- Save 300 µL from each culture for flow cytometry
                            1. Place 300 µL sample in 4 °C refrigerator to halt growth- Repeat steps 6-9 four more times to generate time points every hour for five hours- 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- BBa\_K584000 Culture Dilution
            1. (1.5 OD) (x mL) = (0.05 OD) (20 mL)
                x = 0.667 mL = 667 L original culture

Glycerol Stock

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

LB Broth Preparation

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

TB Broth Preparation

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

Polymerase Chain Reaction (PCR)

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

Gibson Cloning

1. Materials
   1. Autoclaved nanowater- NEBuilder HiFi DNA Assembly Master Mix- PCR tubes- Chemically-competent cells- SOC media- Chloramphenicol plates- Culture beads- 37 °C incubator- 37 °C shaker- Hot water bath- Ice bath- Thermocycler- Procedure
     1. Obtain parts, vector, nanowater, master mix, and PCR tubes
        1. Thaw and vortex master mix thoroughly and keep on ice- 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)- Dilute each part with 10 µL of autoclaved nanowater to make an approximate concentration of 100 ng/µL- Add the following quantities to the PCR tube (total of 20 µL):
              1. Insert: x- Vector: x- Autoclaved nanowater: x- HiFi DNA Assembly Master Mix: 10 µL- Incubate samples in a thermocycler at 50°C for 60 minutes. Once completed, store on ice or at -20°C- Once completed, use right away or store at -20°C. Thaw chemically-competent cells on ice when ready to use the samples.- 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.- Place the mixture on ice for 30 minutes. Do not mix.- Heat shock at 42°C for 30 seconds. Do not mix.- Transfer tubes to ice for 2 minutes- Add 950 µL of room-temperature SOC media to the tube- Incubate the tube at 37°C for 60 minutes. Shake vigorously (250 rpm)- Warm chloramphenicol plates to 37°C- Add 100 µL of cells to the plate, and use beads to spread out the cells- 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

Sequencing

1. Materials
   1. DNA sample- Sequencing tube- 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)- 4 µL of 2 µM Forward Primer- Autoclaved Nanowater(8µL - volume of DNA)- Send tube to Eurofin for sequencing

Tryptone Agar (1% tryptone, 0.25% agar)

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

Miniprep (QIAprep)

1. Materials
   1. Qiagen Resuspension Buffer (Buffer P1)- Qiagen Lysis Buffer (Buffer P2)- Qiagen Neutralization Buffer (Buffer P3)- Qiagen Neutralization Buffer II (Buffer N3)- Qiagen Binding Buffer (Buffer PB)- Qiagen Wash Buffer (Buffer PE)- 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).- Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.- 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.- 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.- Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.- 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.- Note: This step is only required when using endA+ strains or other bacteria strains with high nuclease activity or carbohydrate content.- 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.- Centrifuge for 1 min to remove residual wash buffer.- 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.- 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.

CCMB Solution

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

Competent Cell Preparation

1. Materials
   1. CCMB Solution- Colony of strain of interest- Procedures
     1. Day One
        1. Inoculate 5mL of LB with a single colony of the strain of interest.- Grow the culture overnight at 37 \* C with shaking.- 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.- Inoculate 100 mL of LB containing appropriate antibiotic with 1.0 mL of the overnight culture.- 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.- 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.- 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.- 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.- 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.- Quickly remove all the supernatant using a pipet.- Completely resuspend the cell pellets in 4 mL of cold CCMB solution.- 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.- Return the CCMB solution back to the fridge for storage.

Motility Characterization

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

Sensing Characterization

1. Materials
   1. BBa\_K1022100 cells- BBa\_K3191101 cells- BBa\_K3191102 cells- BBa\_K206000 cells- L-arabinose- AIP- Autoclaved NanoWater- LB Media- 1x PBS- Centrifuge- Plastic culture tubes- Plate reader- Flow cytometer- Procedure
     1. Remove inoculated cultures from shaker and test the OD600 using 100 µL and the plate reader.- 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- 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- 20% arabinose: 5 µL- AIP:
                 1. AIP: 4 µL- 20% arabinose: 0 µL- arabinose:
                   1. AIP: 0 µL- 20% arabinose: 5 µL- Neither:
                     1. AIP: 0 µL- 20% arabinose: 0 µL- Remove 300 µL from each of the cultures; these is the hour 0 samples- Place the cultures in the shaker at 250 rpm and 37 °C for 8 hours- 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- Use plastic culture tubes- Discard supernatant- Add 300 µL of PBS and resuspend- 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- 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- Use plastic culture tubes- Discard supernatant- Add 300 µL of PBS and resuspend- 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- Save the rest of the cultures for flow cytometry
                          1. Place samples in 4 °C refrigerator to halt growth- Place cultures on ice and transport to flow cytometry lab- Calculations
       1. AIP:
          1. (2500 µM)(v) = (10 µM)(1000 µL) v = 4 µL- Arabinose:
            1. (20%)(v) = (0.2%)(1000 µL) v = 10 µL

Killing Characterization

1. Materials
   1. 1 L M9 broth- Three 1 L Erlenmeyer flasks- Rotovap- 37 °C shaker- Dry ice- Acetone- Ethyl acetate- 25 or 50 mL round bottom flask- 20% L-arabinose sterile solution- Kanamycin (50 µg/mL)- 50 mL sterile conical tubes- Autoclaved nanowater- 50% acetonitrile, 5% formic acid, 45% H2O solution- Inoculating loops- Plastic culture tubes- Procedures
     1. Prepare culture tubes with 5 mL M9 broth, 5 μL kanamycin- Inoculate GarKS strains and pSB3K3 negative control from glycerol stock into culture tubes- 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.- Place the entire culture into a 1 L flask containing 250 mL of M9 broth- Incubate the large flasks in a 37 °C shaker at 250 rpm until OD reaches 0.5- Induce with L-arabinose to create a 0.2% concentration in culture medium- Allow cells to grow to OD of 2.0 following induction of L-arabinose- Pour the cell culture into a 250 mL centrifuge bottle- Harvest cells by centrifugation at 5500 rpm for 10 minutes at 4 °C- Pour the supernatant into new 250 mL centrifuge bottle- Use 5 mL of supernatant to resuspend the cell pellet in the original bottle- Transfer pellet resuspension to a 50 mL conical tube- Centrifuge the 50 mL conical tube at 5500 rpm for 10 minutes at 4 °C- Pour the supernatant back into the 250 mL centrifuge bottle in step 10- Store the cell pellet at -80 °C and store the supernatant at 4 °C for later use- Pour the supernatant into a 1 L round bottom flask- Rotovap the supernatant to decrease the volume to approximately 7 mL- Pour the supernatant into a new 50 mL conical tube- 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- Store the conical tube at -80 °C for future analysis- Thaw the supernatant in the 50 mL conical tube and aliquot 3 mL into a 25 mL or 50 mL round bottom flask- Rotovap to dryness- Create 10 mL of the 50% acetonitrile, 5% formic acid, 45% H20 solution- Add 500 µL of acetonitrile/formic acid solution to dissolve the protein- Incubate at room temperature for 1 hour; store at 4 °C for Bradford assay or other analysis

Bradford Assay

1. Materials
   1. Bio-Rad protein assay- 96 well plate- Plate reader- Procedure
     1. Prepare the following standard solutions with bovine serum albumin (BSA) and autoclaved nanowater:
        1. 0 mg/mL BSA- 0.125 mg/mL BSA- 0.25 mg/mL BSA- 0.50 mg/mL BSA- 0.75 mg/mL BSA- 1.0 mg/mL BSA- Dilute the protein sample with autoclaved nanowater to create the following dilutions:
          1. 1:30 dilution- 1:20 dilution- 1:10 dilution- 5:5 dilution- 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- 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.- After 10 minutes, place the 96 well plate in a plate reader to measure the OD at 595 nm- Use the absorbance values for the BSA standards to generate a standard curve of absorbance vs. concentration- 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- Use the dilution values to calculate the concentration of protein in the undiluted sample

20% L-arabinose Preparation

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

M9 Salts Preparation (500 mL)

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

M9 Broth Preparation (1 L)

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

Ligation Cloning

1. Materials
   1. T4 buffer- T4 ligase- Insert- Vector- 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- Plasmid size: \_\_\_\_\_\_\_\_\_\_\_\_ bp – insert size=\_\_\_\_\_\_\_\_\_\_\_\_\_\_ bp- 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- 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- 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- 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- Prepare the following mix for reaction for 10μL
                  1. T4 buffer 1μL- T4 ligase 0.5μL- Insert x- Vector x- Autoclaved Nanowater x

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#

* [Results](#section1)
* [Sensing](#section2)
* [Motility](#section3)
* [Killing](#section4)

# Results

Below are the results of the characterization of the three mechanisms that make up our system: sensing, motility, and killing.

# Sensing

**Purpose:**

We constructed a new BioBrick for the *agr* system, BBa\_K3191101, that includes a sequence modification of an existing *agr* BioBrick: BBa\_K1022100. We first conducted experiments to find if BBa\_K3191101 function is improved when compared to BBa\_K1022100. Two experimental protocols were followed: the first, designated as pre-permeabilized, followed the procedure used in the original characterization of BBa\_K1022100. The second, designated as permeabilized, used a Tris-EDTA solution to permeabilize the outer membrane of the *E. coli* cells.

The native *agr* system contains SarA, which is a transcriptional regulator believed to activate P2, increasing transcription in the *agr* system. To determine if SarA improves P2 transcription, we conducted a second set of experiments adding SarA to BBa\_K3191101. The new construct, BBa\_K3191102, contains the same sequence as BBa\_K3191101 with the addition of the SarA gene.

**Results:**

![](https://static.igem.org/mediawiki/2019/0/07/T--UNebraska-Lincoln--007.jpg)

**Figure 1.** Total fluorescence of pre-permeabilized cells with and without AIP induction.

![alt_text](https://static.igem.org/mediawiki/2019/9/98/T--UNebraska-Lincoln--006.jpg "image_tooltip")

**Figure 2.** Total fluorescence of permeabilized cells at 0 and 3 hours post-induction with AIP.

![alt_text](https://static.igem.org/mediawiki/2019/6/6e/T--UNebraska-Lincoln--009.jpg "image_tooltip")

**Figure 3.** Total change in fluorescence of permeabilized cells with and without AIP induction.

**Discussion:**

Our characterization data displays the difference in fluorescence values between BBa\_K206000, BBa\_K1022100, BBa\_K3191101, and BBa\_K3191102. This data was analyzed with a two-tail t-test of unequal variances with a significance level of 0.05 to draw conclusions of significance. Based on these values, fluorescence for BBa\_K1022100 and BBa\_K3191101 tend to exceed the control. This demonstrates that BBa\_K1022100 and BBa\_K3191101 both function properly.

Figure 1 demonstrates fluorescence values under the pre-permeabilization protocol. These data indicate that AIP induction caused minimal difference in fluorescence intensity. Figures 2 and 3 show fluorescence intensity and change in fluorescence, respectively, of cells after the permeabilization protocol. When comparing total fluorescence, BBa\_K1022100 tends to yield significantly greater fluorescence values than BBa\_K3191101 at hour 8, as supported by a p-values of 0.002 for the AIP trial. This suggests the modified sequences, while more complete, do not successfully increase GFP production under the original characterization procedures. The addition of SarA also does not appear to provide a significantly greater fluorescence under the influence of AIP. This is seen in a comparison of BBa\_K3191101 and BBa\_K3191102 at hour 3 in figure 4 (p-value = 0.902).

Trends in the data suggest that the introduction of AIP appears to have a greater effect on BBa\_K3191101 and BBa\_K3191102 than on BBa\_K1022100 under the permeabilization characterization conditions. The fluorescence values for these conditions are seen in figures 2 and 3. In these conditions, a Tris-EDTA solution is used to attempt to increase AIP intake by the cells. BBa\_K1022100 does not experience significantly greater fluorescence than the control at hour 3 with AIP induction (p-value = 0.111). In comparison, BBa\_K3191101 and BBa\_K3191102 have significantly greater fluorescence at hour 3 with AIP induction. This is demonstrated with p-values of 0.010 for BBa\_K3191101 and 0.002 for BBa\_K3191102. This suggests that AIP induction does not significantly increase fluorescence in BBa\_K1022100, while it does increase in BBa\_K3191101 and BBa\_K3191102.

The increase in fluorescence may be explained by the modified experimental procedure. If the presence of Tris-EDTA successfully increases AIP uptake, then an increase in AIP uptake only improves fluorescence in BBa\_K3191101 and BBa\_K3191102. This suggests that our parts are truly induced by AIP, while BBa\_K1022100 is not.

# CheZ Motility

Purpose: CheZ directed motility allows for the controlled movement of cells in response to a chemical stimulus. Overexpression of CheZ has been noted to constrict overall movement, therefore our system utilizes a degron sequence, YbaQ, to control this phenomenon. Degron sequences tag a protein for degradation, preventing CheZ overexpression in our system. The CheZ-YbaQ sequence is placed under the control of a P2 promoter controlled by the *agr* system as a means to encourage movement towards AIP.

**Results:**

![](https://static.igem.org/mediawiki/2019/a/af/T--UNebraska-Lincoln--011.jpg)

**Figure 1.** Average area of diffusion assays under varying arabinose concentrations without the YbaQ degron.

![alt_text](https://static.igem.org/mediawiki/2019/b/b0/T--UNebraska-Lincoln--010.jpg "image_tooltip")

**Figure 2.** Average area of diffusion assays with the YbaQ degron under varying arabinose concentrations.

After characterization, we concluded our degron was successful, however overall motility failed.

The strain UU2685 *E. coli* cells without the transformed CheZ system exhibited the expected lack of motility. The cells transformed with our CheZ parts, pBAD+CheZ and pBAD+CheZ+YbaQ displayed significant motility, even without the induction of arabinose.

The pBAD+CheZ showed consistent diffusion motility for both 0% and 0.02% induction. A two-tail t-test of unequal variances with a significance level of 0.05 displayed a p-value of 0.092, indicating a lack of significance in the area of diffusion. This contradicts the hypothesis of pBAD+CheZ relying on arabinose induction for motility. The reasoning behind this lack of significance is inconclusive, but may be a result of arabinose contamination from an unaccounted source such as the cell media. An additional possibility is our overestimation of promoter regulation. The pBAD+CheZ induced with 0.2% arabinose displayed motility significantly different (p-value of 0.005) from the combined motility of uninduced pBAD+CheZ and pBAD+CheZ induced with 0.02% arabinose. It was concluded this was likely a result of the greater concentration of arabinose providing the cells with more sugar to consume over time, permitting a greater displacement.

![alt_text](https://static.igem.org/mediawiki/2019/0/0d/T--UNebraska-Lincoln--pbadchez021.jpg "image_tooltip")

**Figure 3.** Motility of pBAD+CheZ construct with 0.2% arabinose.

![alt_text](https://static.igem.org/mediawiki/2019/9/9a/T--UNebraska-Lincoln--pbadchez0021.jpg "image_tooltip")

**Figure 4.** Motility of pBAD+CheZ construct with 0.02% arabinose.

![alt_text](https://static.igem.org/mediawiki/2019/2/2b/T--UNebraska-Lincoln--pbadchez01.jpg "image_tooltip")

**Figure 5.** Motility of pBAD+CheZ construct with 0% arabinose.

The pBAD+CheZ+YbaQ induced with 0.02% arabinose produced an area of diffusion comparable to that of pBAD+CheZ+YbaQ when induced with 0% arabinose. A two-tail t-test of unequal variances with a significance level of 0.05 displayed a p-value of 0.978, indicating a lack of significant difference. The lack of significance can be attributed to similar conditions discussed in the pBAD+CheZ analysis when the same trend was observed. The pBAD+CheZ+YbaQ induced with 0.2% arabinose was significantly more motile than the motilities of pBAD+CheZ+YbaQ with no induction and 0.02% induction combined. This was maintained by a p-value of 0.0039 generated by a t-test similar to the one mentioned above.

![alt_text](https://static.igem.org/mediawiki/2019/a/af/T--UNebraska-Lincoln--pbadchezybaq002.jpg "image_tooltip")

**Figure 6.** Motility of pBAD+CheZ+YbaQ construct with 0.2% arabinose.

![alt_text](https://static.igem.org/mediawiki/2019/9/9a/T--UNebraska-Lincoln--pbadchez0021.jpg "image_tooltip")

**Figure 7.** Motility of pBAD+CheZ+YbaQ construct with 0.02% arabinose.

![alt_text](https://static.igem.org/mediawiki/2019/2/2b/T--UNebraska-Lincoln--pbadchez01.jpg "image_tooltip")

**Figure 8.** Motility of pBAD+CheZ+YbaQ construct with 0% arabinose.

The motility observed for the pBAD+CheZ construct with no arabinose induction was registered as a false positive; the lack of arabinose should have yielded no motility. This implies the presence of background CheZ. However, the pBAD+CheZ+YbaQ displayed significantly less motility (two-tail t-test p-value of 0.02) indicating a decrease in the CheZ protein with the presence of the degron. We have concluded this indicates the degron is successful in degrading excess CheZ to allow for the efficient function of chemotaxis. The inhibition of excess CheZ is imperative to the success of the motility mechanism because it ensures that MRSA is precisely targeted as intended. Under optimal conditions (an induction of 0.2% arabinose), pBAD+CheZ showed no significant difference (p-value of 0.766) in motility compared to pBAD+CheZ+YbaQ.

While there was significant diffusion of the CheZ parts as detailed above, there were no indicators of successful directionality. The cells proliferated in a circular region that contested any form of directed motility.

P2+CheZ+YbaQ was never successfully sequenced and therefore never characterized. It remains unknown how the P2 promoter would have reacted in the context of our sensing system.

# Killing

Garvicin KS

Purpose: The small antimicrobial peptide (AMPS) Garvicin is the final step in our 3-modular system. Although newly discovered, this multi-peptular bacteriocin has been noted to be more potent than many other bacteriocins. Once the motility function has carried the system to the desired Staph. Aureus, the payload , garvicin KS, will be released into the infested site. As *E. coli* is a Gram-negative bacterium, additional steps must be taken to ensure that the garvicin KS peptides can be secreted from the cell. Using a novel signal peptide (NSP4) this peptide tags proteins for secretion by the Sec pathway allowing for greater production of Garvicin into the system.

We are testing the effectiveness of our garvicin part through Bradford analysis and comparing its concentration to a negative control, garvicin alone and garvicin with the NSP4 secretion tag. It is our prediction that garvicin with the NSP4 should be secreted at a higher concentration.

**Results:**

![](https://static.igem.org/mediawiki/2019/e/ee/T--UNebraska-Lincoln--013.jpg)

**Figure 1.** Protein concentration obtained from cell supernatant measured in Bradford assay.

![alt_text](https://static.igem.org/mediawiki/2019/0/05/T--UNebraska-Lincoln--014.jpg "image_tooltip")

**Figure 2.** Protein sample measured in Tris-Tricine gel. The red bar indicates the expected location of garvicin KS.

**Discussion:**

The concentrations of protein collected from a Bradford assay can be seen in figure 1. The negative control, pSB3K3, had the lowest noted concentration of 0.335 mg/mL. This indicates the basal level of protein production in the cells. The culture containing the garvicin KS gene displayed a protein concentration of 0.528 mg/mL, indicating an increase in the concentration of protein produced and excreted from the cell. The culture expressing garvicin with the NSP4 secretion peptide demonstrated a concentration of >1.044 mg/mL. This suggests that the inclusion of the secretion peptide successfully increased protein secretion.

The results of the Mini-Protean Tris-Tricine gel, which separates proteins by size similar to SDS-PAGE, revealed that all three samples might contain some level of protein contamination. This will interfere with the identification of the target protein. While there are bands present at the size that corresponds to the garvicin KS peptide, the blur associated with possible contamination of the sample interferes with the identification. Mass spectrometry analysis can be conducted on the protein sample to more conclusively identify the molecular weight of the protein.

The group had plans to include the regulatory promoter, P2, in placement of the pBAD promoter utilized in each garvicin part. The intentions of the group was that P2 would provide greater activation than the pBAD itself, however time constraints disallowed this inclusion.

#### Address

University of Nebraska-Lincoln iGEM Team

634 Hamilton Hall

Lincoln, NE 68588-0304

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