Team:Harvard BioDesign/Experiments

Harvard BioDesign 2016

Experiments

For the outcome of these experiments, please visit our results page.

Experiments: Protein Characterization

Experiment name: sfGFP visualization

What experiment will show: This experiment visualizes the sfGFP reporter protein in our PETase-sfGFP fused protein. It shows that our cells are expressing our fused protein.

Reason for doing experiment: Validates PETase-sfGFP expression and characterizes the PETase-sfGFP part

Experiment methods: Grew up T7 lysY Iq cells containing our plasmid. Induced with IPTG following the protocol for “Expression of T7 Cells” in general protocols below. Visualized cells using a Zeiss Axio Observer.Z1 fluorescent microscope and took pictures (see results page).

Controls: Also used an uninduced sample.

Experiment name: SDS-PAGE

What experiment will show: An SDS page separates the proteins in a sample by weight. We will be looking for a protein band at the expected size for PETase.

Reason for doing experiment: Running an SDS page will show us whether our protein is being expressed in our cells.

Materials: 5x Tris-Glycine SDS Running Buffer, Mini-PROTEAN® TGX Stain-Free™ Precast Gels, Coomassie Blue Stain

Experiment methods: After growing up expression cells and inducing them with IPTG, we then lysed the cells using either ultrasonication or freeze-thaw (see our general lab protocols below for more information). The cell lysate including supernatant was then run on an SDS page. We followed the Mini-PROTEAN® Precast Gels Instruction Manual and Application Guide for gel electrophoresis.

Controls: For our controls, we used cells that contained the same construct, but were not induced.

Experiment name: Western blot

What experiment will show: A Western blot detects whether a specific protein is present by using antibodies or other binding methods.

Reason for doing experiment: We can validate the presence of our protein using a Western blot. Usually a western blot uses a specific antibody to bind the target protein. However, since our protein is newly discovered and there have not been any antibodies found for it, we included in the design of our PETase construct a histag. The histag is a sequence of six histidine amino acids that has a commercially-available antibody. By performing a Western blot we will observe only proteins with histags (and the only protein in our cells with a histag should be our recombinant protein).

Materials: Novex NuPAGE® precast gels, NuPAGE® Antioxidant, NuPAGE® LDS Sample buffer 4x, NuPAGE® Sample Reducing Agent 10x, MES Running buffer, Novex PreStained Protein Ladder, MagicMark™, TBST, Dry milk, horseradish peroxidase antibody, XCell SureLock™ Mini-Cell Electrophoresis System, iBlot® 7-Minute Blotting System (corresponding Novex kit containing blotting paper, transfer membrane, iBlot™ Gel Transfer Stacks)

Experiment methods: Before performing a Western blot we had to do a histag purification (see general lab protocols below) Afterwards, we followed the following protocol:

1) Sample Preparation:

    Reduced Samples:
  • Sample: 5µl
  • NuPAGE LDS Sample Buffer (4x): 2.5µl
  • NuPAGE Reducing Agent (10x): 1µl
  • Deionized Water: 1.5µl

    Nonreduced Samples:
  • Sample: 5µl
  • NuPAGE LDS Sample Buffer (4x): 2.5µl
  • Deionized Water: 2.5µl

2) Heat samples for 10 minutes at 70 degrees Celsius.

3) Prepare ladder (5µl of Novex Pre-Stained Protein Ladder and 5µl MagicMark).

4) Prepare electrophoresis apparatus (and precast gels) in accordance with manufacturer's instructions.

5) Add MES running buffer filling both chambers to the top.

6) Add 500µl of Antioxidant to the upper chamber.

7) Load 10µl of ladder and each sample into the wells.

8) Run gel at 200V for 35 minutes.

9) Refer to Thermo Fisher device manual for iBlot® 2 Dry Blotting System instructions (pages 21-25); select “p0” template method.

Controls: For our controls we used uninduced cells.

Link to results page

Experiment name: p-nitrophenyl butyrate (pNPB) Assay

What experiment will show: This experiment will show whether our enzyme is catalytically active. p-nitrophenyl butyrate (pNPB) is a substrate that contains an ester bond resembling the ester bond broken in PET degradation. PETase, and other PET-degrading enzymes are expected to break the ester bond in pNPB. When the bond is broken, the resulting product emits a wavelength of 405nm that can be detected by a plate reader set to read absorbances at 405nm. If our enzyme is catalytically active, we expect this experiment to show an increase of absorbance at 405nm after adding pNPB substrate.

Reason for doing experiment: To show that our enzyme is catalytically active.

Materials: As adapted from 2012 UC Davis iGEM’s LC Cutinase pNPB assay: pNPB buffer (should be made and used in fumehood due to acetonitriles toxicity)

  • 10mM pNPB in acetonitrile
  • 1:4:95 ratio of acetonitrile pNPB solution, 100% ethanol and 50mM Tris-HCL (pH 8)
  • Plate reader
  • 24 well plate
  • Cell lysate

Experiment methods: Set up 24 well plate by adding cell lysate and controls. Add the pNPB buffer immediately before running the plate reader. Run the plate at 37C for 10mins inside the plate reader, taking measurements of 405nm absorbance every 15 seconds. 405nm is the absorbance of pNPB substrate that has been broken by an esterase enzyme, so a significant read at 405nm absorbance indicates enzyme is breaking ester bonds in the substrate.

Controls: For each induced cell lysate sample, we added either lysis buffer (no pNPB substrate) or pNPB buffer. Additionally, we had uninduced controls with pNPB buffer, and a blank well with only pNPB buffer.

Experiment name: pH optimization for PETase E. coli

What experiment will show: How well PETase E. coli grows at different pHs

Reason for doing experiment: PETase is most active at higher pHs, so we want to determine a happy pH medium between E. Coli growth and enzyme activity.

Materials: NaOH, E. Coli, LB

Experiment methods: We set up triplicates in 48 well plates of E. coli with lb set to different pHs and left it in an overnight plate reader for 350 mins.

Controls: We also ran triplicates of just LB (without E. coli) but because this was not very useful information, we didn’t include it in the results page.

Experiments: Microbial Fuel Cell

Experiment name: D. tsuruhatensis Metabolizing Terephthalic Acid (TPA)

What experiment will show: Whether D. tsuruhatensis can grow using TPA as its sole carbon source

Reason for doing experiment: In order to use D. tsuruhatensis in a microbial fuel cell with TPA as the carbon source, the bacteria has to be able to efficiently metabolize TPA.

Materials: TPA, M9 Minimal Media, D. tsuruhatensis, Well Plates, Optical Density (OD) plate reader.

Experiment methods: Prepare plates that contain the following: 1. Just M9 Medium 2. M9 + TPA 3. M9 + TPA + D. tsuruhatensis 4. M9 +D. tsuruhatensis. *Plates were grown for just over 14 hours at 30 degrees celsius.

Controls: Plates containing M9 Medium alone; plates with M9 media and TPA; plates with M9 media and D. tsuruhatensis

Experiment name: Voltage Measurement in the Fuel Cell

What experiment will show: Will show whether injecting TPA into our microbial fuel cell with Delfita and methylene blue will result in an increase in voltage.

Reason for doing experiment: This will signify that our bacteria can use TPA as a substrate to produce electricity, and thus a signal.

Materials: Fuel Cell with D. tsuruhatensis, TPA, M9 Minimal Media, D. tsuruhatensis Broth, Methylene Blue, Voltmeter connected to a PC

Experiment methods: We first centrifuge the D. tsuruhatensis bacteria into a pellet in order to remove it from the original broth (which has a carbon source). We then resuspend the D. tsuruhatensis in M9 minimal salt medium (which doesn’t have a carbon source), then we add methylene blue and add it to the fuel cell. After the voltage has plateaued, we add TPA to the mix to see whether there is an increase in voltage.

An electrometer that could be connected to a PC was purchased and is set up in a way that the black rod’s metal tip (negative) is constantly touching the anode, while red rod’s tip (positive) touches in on the cathode. Not only voltage with respect to time can be detected this way, but also the current can be measured in series and readings in the milliamps range should be expected.

First, before adding the liquid medium or bacteria inside the cell, the resistance is checked and it should be in the Mega or Kilo Ohms range (if not, the electrodes are touching touching the bolts and there is a need to reassemble).

Controls: Bacteria in M9 Media and methylene blue inside the fuel cell, with no TPA

Experiment name: Selecting Exogenous Mediator Type and Concentration

What experiment will show: Will show which exogenous mediator type and concentration has the biggest color change.

Reason for doing experiment: Exogenous mediators are essentially water-soluble redox molecules that can shuttle electrons from the microbe to the anode and therefore allowed us to use microbes without electron shuttling membrane proteins in the fuel cell. This experiment will show us which exogenous mediator type and concentration is the best for the microbial fuel cell.

Materials: Methylene blue, neutral red, C2566H, C2530H, M9 minimal medium

Experiment methods: We tested two strains of E. Coli, with two different types of exogenous mediators (methylene blue and neutral) at 6 different concentrations (0.1, 0.05, 0.01, 0.005, 0.001, 0.0005). This would determine which exogenous mediator type and concentration is the best with a bacteria known to work in a microbial fuel cell (using the mediator method rather than the membrane protein method).

We decided to test methylene blue and neutral red as exogenous mediators because The 2013 Bielefeld iGem team worked on a similar experiment and these two mediators worked best for them. (iGem Bielefeld, 2013).

Based on the Bielefeld team’s experimental work, we conducted an experiment with our E.Coli and the exogenous mediators to see if they were able to reduce these. The overnight reduction of methylene blue and neutral red, which is visually obvious from the color change but also checked with a plate reader, comes from the bacteria’s ability to reduce the chemicals appropriately. Based on Dr. Parra suggestions and its widespread use, methylene blue was used as the exogenous mediator in all future experiments after we confirmed that our strains were able to reduce it.

Controls: Exogenous mediator and M9 medium without bacteria.

Experiments: General Lab Protocols

Experiment name: Freeze Thaw

Materials: Liquid culture of cells

Experiment methods:

Protocol obtained from European Molecular Biology Laboratory:

1) Freeze quickly on dry ice and leave for 3 min.
2) Thaw immediately at 42°C. Vortex vigorously to mix well.
3) Repeat the two previous steps for three more times (4 freeze-thaw-vortex cycles in all).

Experiment name: Ultrasonication

Materials:

  • 250mL flask of expression cell (Arabinose + K936020 composite on 6/14; transformed by head shock into non-T7expression strain)
  • Lysis Buffer
  • 20x TBS (Tris-HCl & NaCL)
  • 1 tablet of PMSF (1/15mL) - protease inhibitor
  • 1 uL benzoase (DNAase) [yellow box in the freezer - white cap]

Experiment methods:

Preparation
1) Stock Lysis buffer: Prepare lysis buffer by diluting 20x TBS (500mL) with 1L H2O (50mL/L)
2) Filter the lysis buffer w/ Corning 1L filter system
3) store in the freezer
Procedure
4) Keep the suspension at all times on ice
5) Add 1 PMSF tablet & 1uL benzoase to 25mL lysis buffer. Vortex.
6) Centrifuge the cells: 15 min, 4-5000G. Label as supernatant as S1 and keep in the fridge.
7) Resuspend the cells in 25mL lysis buffer
8) Cool the cell suspension on ice for 10 min
9) Sonicate the cell suspension w/ following settings:
Amplitude: 40
Process Time: 3:00
Pulse-ON Time: 0:30
Pulse-OFF Time: 0:30
Note: spray & wipe down w/ 70% ethanol before & after use. Tip should NOT touch the tube.
10) Remove all cell debris by ultracentrifugation at 4°C for 30 min at 15,000-20,000G. Label the supernatant as S2 and keep in fridge. Save 1mL lysate for SDS-Page. Store pellet in the freezer

Experiment name: Histag purification

In purification techniques, the histidine residues are able to bind to immobilized Nickel ions, and can then be visualized.

Materials:

Experiment methods:
1) Set up columns: create hole on top of 14mL Falcon tube, put in column, filter, & cap)
2) Add agarose beads to column
3) Let ethanol drain from beads
4) Wash beads with 2mL of buffer PB (x3); wait for it to filter through column between each time
5) Cap bottom of column
6) Save 1mL lysate for SDS-PAGE. Add the rest of supernatant and resuspend (Put back in tube of supernatant and continue resuspending)
7) Put in flip shaker for 2hrs (can potentially prepare SDS-PAGE during this time)
8) Put sample back into column, keep flowthrough
9) Elute (different concentrations of imidazol + phosphate buffer)
10) Concentrate elute & supernatant in 10kDa Amicon (Next time, try 25kDA Amicon) - centrifuge 30min @4000g
11) Move concentrate to smaller tubes
12) Keep flowthrough of the concentrate

Experiment name: Expression of T7 Cells

Materials: NEB T7 Express lysY/Iq Competent E. coli (High Efficiency), IPTG, Carbenicillin, LB media

Experiment methods:

Note: Inoculate at least 2 colonies per plate. Since they are from the same plate, they contain the same plasmid. When inducing with IPTG, add IPTG to only one of the duplicates. This means we will have an induced and uninduced condition for the same plasmid (1 control for each induction).

1) Transform expression plasmid into NEB Express Iq. Plate on ampicillin selection plates and incubate overnight at 37°C.

2) Resuspend a single colony in 10 ml liquid culture with ampicillin and glucose:

  • 8 mL LB
  • 2 mL of Glucose (10% stock) for final glucose concentration of 2%
  • 10uL carbenicillin
  • Remember to do two colonies per condition, so take two colonies from the same plate.

3) Incubate at 37°C until OD600 reaches 0.4 - 0.6.

  • When checking OD, take 100 uL of culture and add to 900 uL of LB media in an OD cuvette. Measure OD of dilution and multiply the OD600 by 10 to get the approximate OD. (You can check 1mL of culture when you believe the approximation is close to .5).
  • Since cells grow fast you may have to re - inoculate before inducing with IPTG. Inoculate (8mL LB, 2 mL Glucose stock, antibiotic) fresh culture with overnight culture. Remember to OD before inducing.

4) After reaching proper OD, spin down cells and discard supernatant (it has glucose in it). 4000g for 5 mins should work. Resuspend pellet in 10mL LB.

5) Induce with IPTG:

  • 4 µl of a 1M stock of IPTG if doing 10mL culture (final concentration of 400 µM)
  • Induce for 6 hours at 37°C.
  • Remember to induce only one of your duplicates and keep the other as an uninduced control.

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