Difference between revisions of "Team:Baltimore Bio-Crew/Experiments"

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<button class="dates">Ni-NTA Spin Column Purification of 6xHis-Tagged Proteins (E. coli Lysates)</button>
 
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   <p>Math:</p>
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    <li>Resuspend a pellet derived from 5ml cell culture volume in 630 ul Lysis buffer (NPl-10). Add 70ul Lysozyme Stock Solution (10mg/ml) and add 3 units/ml culture volume Benzonase Nuclease</li>
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    <li>Incubate for 15-30min</li>
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    <li>Centrifuge lysate at 12000 x g for 15-30 min at 4 degrees C. Collect the supernatant.</li>
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    <li>Save 20 ul of the lysate for SDS-PAGE analysis</li>
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     <li>Equilibrate the Ni-NTA spin column with 600 ul buffer NPl-10. Centrifuge for 2 minutes at 2900 rpm. Spin without lid to ensure it is done in two minutes</li>
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     <li>Load up to 600 ul of the cleared lysate containing the His-Tagged protein onto the (pre-equilibrated) Ni-NTA spin columns. Spin for 5 minutes at about 1600 rpm, and collect the flow-through. To ensure efficient binding, don’t go higher than 1600 rpm, though do it with an open lid</li>
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     <li>Wash the column twice with 600 ul Buffer NPl-20. Centrifuge for 2 min. at about 2900 rpm. Save the flow-through for analysis by SDS-PAGE</li>
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    <li>Elute the protein twice with 300ul Buffer NPl-500. Centrifuge for 2 minutes at about 2900, and collect the eluate. Most of the protein will be in the first eluate. The rest will be in the second. 300ul each time</li>
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Revision as of 14:10, 31 October 2017


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Experiment


Overview


We are evaluating our parts by verifying that they can be used to degrade PET plastic. We are setting up an experiment in which we will test for the ability of E.coli bacteria engineered with each part to degrade PET plastic. Glass culture tubes were set up containing LB media and small squares of PET plastic that had been weighed beforehand. The approximate dimensions of the plastic squares were 20x15x0.5mm, and they weighed between 101 and 89 mg. After the bacteria with the gene have been added, the plastic will be weighed twice a week to check for lost material. Some tubes will be incubated at 30 degrees Celsius because that is the best growth temperature for the Ideonealla bacteria, and some will be incubated at 37 degrees Celsius because that is the best temperature for E. coli.


Protocols

Adapted from Addgene, (https://www.addgene.org/protocols/gel-electrophoresis/)

Pouring the Gel

  • Measure out agarose on scale slowly (for a standard 1% gel, use 1 g of agarose. For other concentrations, calculate accordingly). Stay within ~.05 g of 1 g.
  • Mix agarose powder with 100 mL 1xTAE in a microwavable flask.
  • Microwave for 1-3 min until the agarose is completely dissolved, stopping every 30 seconds to swirl the flask. Keep an eye on it to make sure it does not overboil. Wear oven mitts when handling the flask.
  • Place agarose solution on counter until the flask can be comfortably held
  • Set up the gel tray with the well comb in place, as one person slowly pours the agarose solution into the well. Avoid bubbles.
  • Let gel sit for 20-30 minutes until completely solidified (the gel will be milky white and opaque).

Loading Samples

  • Add equal amount of loading buffer to each of your samples.
  • Place the agarose gel into the electrophoresis unit.
  • Fill gel box with 1xTAE until the gel is covered and container is filled.
  • Carefully load a molecular weight ladder into the first lane of the gel. Place your elbow on the counter to prop yourself up so you can look at the gel from an angle to see the wells. Place the tip of the pipette tip just above the well and slowly push so the sample fills the entire well.
  • Load the rest of the samples in this manner.
  • Run the gel at 80-150 V until the dye line is approximately 75-80% of the way down the gel for about 1 hour. Note: Black is negative, red is positive. Always Run to Red.
  • Turn OFF power, disconnect the electrodes from the power source, and then carefully remove the gel from the gel box.
  • Visualize your DNA fragments under UV light.

All plasmid purifications were done using the QIAprep spin mini prep kit protocol.

Master mix:

  1. 76 uL H2O
  2. 20 uL 10x NEB2
  3. 1 uL EcoR1
  4. 1 uL Pst1
  5. 1 uL Dpn1
  6. 1 uL BSA

Mix together:

  • 10 uL Sample DNA
  • 10 uL Master Mix

Mix together:

  • 2 uL plasmid
  • 3.5 uL insert
  • 1 uL T4 Ligase buffer
  • 0.5 uL T4 Ligase
  • 4.5 uL H2O

We used the iGEM Parts Registry Transformation Protocol: https://static.igem.org/mediawiki/parts/6/67/IGEM_Registry_-_Transformation_Protocol.pdf

Changes made: Recovery tubes were made using 500 uL SOC media instead of 200 uL

Master Mix:

  1. 316.8ul dIH20
  2. 44ul Taq Buffer
  3. 22ul foreward primer
  4. 22ul reverse primer
  5. 33ul dNTPs
  6. 2.2ul Taq

Add 1ul DNA to 19ul Master Mix

  1. Resuspend a pellet derived from 5ml cell culture volume in 630 ul Lysis buffer (NPl-10). Add 70ul Lysozyme Stock Solution (10mg/ml) and add 3 units/ml culture volume Benzonase Nuclease
  2. Incubate for 15-30min
  3. Centrifuge lysate at 12000 x g for 15-30 min at 4 degrees C. Collect the supernatant.
  4. Save 20 ul of the lysate for SDS-PAGE analysis
  5. Equilibrate the Ni-NTA spin column with 600 ul buffer NPl-10. Centrifuge for 2 minutes at 2900 rpm. Spin without lid to ensure it is done in two minutes
  6. Load up to 600 ul of the cleared lysate containing the His-Tagged protein onto the (pre-equilibrated) Ni-NTA spin columns. Spin for 5 minutes at about 1600 rpm, and collect the flow-through. To ensure efficient binding, don’t go higher than 1600 rpm, though do it with an open lid
  7. Wash the column twice with 600 ul Buffer NPl-20. Centrifuge for 2 min. at about 2900 rpm. Save the flow-through for analysis by SDS-PAGE
  8. Elute the protein twice with 300ul Buffer NPl-500. Centrifuge for 2 minutes at about 2900, and collect the eluate. Most of the protein will be in the first eluate. The rest will be in the second. 300ul each time

Math:

Math:

  • Worked on getting set up to start math modeling of plastic degradation. Ready to start next week
  • Found possible alternatives to our plastic degrading enzymes
  • Collaboration: College Park mentoring for plastic degrading simulation

Math:

Math:

Math:

  • Worked on getting set up to start math modeling of plastic degradation. Ready to start next week
  • Found possible alternatives to our plastic degrading enzymes
  • Collaboration: College Park mentoring for plastic degrading simulation

Math:

  • Worked on getting set up to start math modeling of plastic degradation. Ready to start next week
  • Found possible alternatives to our plastic degrading enzymes
  • Collaboration: College Park mentoring for plastic degrading simulation

Math:

Math:

  • Worked on getting set up to start math modeling of plastic degradation. Ready to start next week
  • Found possible alternatives to our plastic degrading enzymes
  • Collaboration: College Park mentoring for plastic degrading simulation

Math:

  • Worked on getting set up to start math modeling of plastic degradation. Ready to start next week
  • Found possible alternatives to our plastic degrading enzymes
  • Collaboration: College Park mentoring for plastic degrading simulation

Math:

  • Worked on getting set up to start math modeling of plastic degradation. Ready to start next week
  • Found possible alternatives to our plastic degrading enzymes
  • Collaboration: College Park mentoring for plastic degrading simulation