Team:Baltimore BioCrew/Experiments

Experiments

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

Introduction

Our project was split into two sections: Clot Makers, who focused on the quick coagulation of wounds using an enzyme found in the Russel's Viper's and Desert Horned Viper's venom. Conversely, the Clot Breakers focused on using a shortened version of tPA called Reteplase to decoagulate unordinary clots, such as those that could appear in blood vessels, causing strokes and heart diseases.

Protocol: Clot Makers

Gel electrophoresis

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.

Plasmid Purification

We did these using the QIAprep Spin Mini Prep Kit Protocol:

Restriction Digest

Master mix:

• 76 uL H2O
• 20 uL 10x NEB2
• 1 uL EcoR1
• 1 uL Pst1
• 1 uL Dpn1
• 1 uL BSA

Mix together:

• 10 uL Sample DNA
• 10 uL Master Mix

Ligation

Mix together:

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

Transformations

We used the iGEM Parts Registry Transformation Protocol:

https://static.igem.org/mediawiki/parts/6/67/IGEM_Registry_-_Transformation_Protocol.pdf>

Changes made: We omitted step 13, and used one petri dish rather than two.

Colony PCR

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

Settings:

1 Cycle

1. 95°C - 1min

30 Cycles

1. 95°C - 30s
2. 55°C - 45s
3. 72°C - 45s

1 Cycle

1. 72°C - 2min

Hold at 4°C

Ni-NTA Spin Column Purification of 6xHis-Tagged Proteins (E. coli Lysates)

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

Protein Resuspension and Protein Gel Electrophoresis

1. Resuspend protein sample in a 1:1 ratio of sample to cracking buffer
2. Place resuspended sample in boiling water for 5 minutes
3. Load samples into protein gel
4. Run at 100v
5. Run until the gel is about 75-80% of the way down the gel
6. Remove gel from electrodes and plastic casing
7. Wash gel with purified water about 3 times, agitating the vessel the gel and water are in slightly to wash thoroughly, while not tearing the gel
8. Stain the gel with Simplyblue dye

PCR Amplification

Mix together:

1. 35.4 uL Water
2. 15 uL 5x Phusion Buffer
3. 1 uL 10 mM dNTPs
4. .8 uL 30 mM Forward Primer
5. .8 uL 30 mM Reverse Primer
6. .5 uL enzyme (Phusion taq)
7. 2 uL Template

Settings

1 Cycle

1. 98°C - 30s

35 Cycles

1. 98°C - 10s
2. 55°C - 30s
3. 72°C - 2min

1 Cycle

• 72°C - 10min

Flourocine Diacetate Enzyme Activity Assay

Enzyme activity was assayed using a modification of the fluorocine diacetate assay for hydrolytic enzymes (Gamble and Muriana 2007). 5ul of in vitro transcription/translation product was mixed with 250ul of phospate buffered saline containing 1 mM flourescine diacetate in wells of a Tecan black Tecan micro titer plate. Flouresence generated by cleavage of the acetate groups was measured using a Tecan Genios plate reader with excitation at 485nM and emission at 535 nM.