Protocols

Smell Testing:

Pyrazine

• Add 200 mL of deionized water into a beaker.
• Add 1 milligram of the Pyrazine powder into the beaker.
• Mix well.
• In order to smell the compound, place it upright and cup your hand. Waft the vapor with the cupped hand and smell.

Theobromine

• Add 200 mL of deionized water into a beaker.
• Add the stir bar and turn the knob to an appropriate speed.
• Add 2 grams of Theobromine powder into a beaker.
• Mix well.
• In order to smell the compound, place it upright and cup your hand. Waft the vapor with the cupped hand and smell.

Phenylethylamine

• Add 200 mL of deionized water into a beaker.
• Add the stir bar and turn the knob to an appropriate speed.
• Add 200 mg of Phenylethylamine powder to the beaker.
• Mix well.
• In order to smell the compound, place it upright and cup your hand. Waft the vapor with the cupped hand and smell.

Furaneol

• Add 200 mL of deionized water into a beaker.
• Add the stir bar and turn the knob to an appropriate speed.
• Add 200 mg of Furaneol powder to the beaker.
• Mix well.
• In order to smell the compound, place it upright and cup your hand. Waft the vapor with the cupped hand and smell.

To Create a Full Solution:

Combine all compounds in the same ratio, except keep the amount of water the same.

PCR for vector amplification:

PCR Reaction for a 25 uL reaction:

• In a PCR tube, add 12.5 uL of Phusion Hot Start Flex 2x Master Mix or Q5 High-Fidelity DNA Polymerase
• Add in 1.25 uL each of the forward and reverse primer at 10uM
• Add 20ng of template DNA, so in our case, 4uL at 5ng/uL of PNZ8148
• Adding enough water to get to 25uL (6uL of water)
• Run in the thermocycler with an initial denature at 98C for 30 seconds, then repeat the following cycle 35 times (denature at 98C for 10 seconds, anneal at 56C for 30 seconds, extend at 72C for 90 seconds). At the end of the cycles, hold at 72C for 5 minutes and then drop down to 4C forever.

Gel Electrophoresis

Preparing the gel:

• Weigh out 0.5g of the agarose on a piece of weigh paper. Transfer to an Erlenmeyer flask. Add 50 ml of 1X TAE.
• Microwave flask in 30 second increments until the agarose and buffer mixture is fully dissolved and remove with cooking mittens. Periodically swirl mixture to ensure the mixture is melting evenly.
• Remove the flask of clear agarose and allow it to cool. This will take about 10 minutes. If you are able to grasp the flask comfortably it is cooled enough to pour.
• While the agarose is cooling, place the gel tray into the gel box and add blocker and a comb.
• When the agarose has cooled, add 2 ul of Apex Safe DNA Gel Stain to the melted agarose.
• Swirl the agarose to incorporate the dye and pour the agarose into the gel tray—your gel should be about 1 cm thick.
• Allow at least 20 minutes for the gel to solidify. Once solid, carefully remove the comb and blockers, and place the solidified gel (still on the tray) oriented on the negative side.
• Add enough 1X TAE buffer to completely cover the gel by 1 cm.

Preparing the Sample and running gel:

• On a piece of parafilm, spot 2 ul of DNA loading dye with 10 uL of PCR product and mix for each sample
• Transfer each mixed sample into wells
• Ensure that appropriate DNA ladder is inserted into well
• Run gel at 100V for ~20-30 minutes and view under UV light

Dpn1 Digestion

Overview

• The objective of DpnI digestion is to selectively cleave methylated DNA, specifically recognizing and destroying DNA that was in bacteria. This is commonly used in molecular biology, particularly cloning, for the following purpose:
  • Elimination of Template DNA: After a PCR or mutagenesis reaction, DpnI digestion removes the parental (template) DNA, which is typically methylated if it was propagated in a bacterial strain like E. coli that methylates specific residues.
• After Dpn1-mediated digestion, it is believed that template DNA (methylated) will be cleaved, leaving only the intended PCR product.

Materials:

• 8uL PCR product (DNA)
• 1uL 10x rCutSmart Buffer
• 1 uL Dpn1

Procedure:

• Combine the PCR product, buffer, and Dpn1 enzyme into a 1.5 mL Eppendorf tube.
• Incubate at 37 C for 30 minutes
• Heat at 80 C for 20 minutes

Column Purification

StrataPrep PCR Purification Kit
Materials:

• DNA-binding solution
• Wash buffer
• Microspin cups
• Receptacle tubes (2 ml)
• Microcentrifuge tubes
• Microcentrifuge

Procedure:

• Add a volume of DNA-binding solution equal to the volume of the aqueous portion of the PCR product to the microcentrifuge tube containing the PCR product and mix the two components.
• Using a pipet, transfer the PCR product–DNA-binding-solution mixture to a microspin cup that is seated in a 2-ml receptacle tube. Snap the cap of the 2-ml receptacle tube onto the top of the microspin cup.
• Spin the tube in a microcentrifuge at maximum speed for 30 seconds.
• Open the cap of the 2-ml receptacle tube, remove and retain the microspin cup, and discard the DNA-binding solution.
• Open the cap of the 2-ml receptacle tube and add 750 μl of 1× wash buffer to the microspin cup. Snap the cap of the receptacle tube onto the top of the microspin cup.
• Spin the tube in a microcentrifuge at maximum speed for 30 seconds.
• Open the cap of the 2-ml receptacle tube, remove and retain the microspin cup, and discard the wash buffer.
• Place the microspin cup back in the 2-ml receptacle tube and snap the cap of the receptacle tube onto the microspin cup.
• Spin the tube in a microcentrifuge at maximum speed for 30 seconds. On removal from the centrifuge, make sure that all of the wash buffer is removed from the microspin cup.
• Transfer the microspin cup to a fresh 1.5-ml microcentrifuge tube and discard the 2-ml receptacle tube.
• Add 50 μl of elution buffer directly onto the top of the fiber matrix at the bottom of the microspin cup. Avoid touching the fiber matrix with the pipet tip.
• Incubate the tube at room temperature for 5 minutes.
• Snap the cap of the 1.5-ml microcentrifuge tube onto the microspin cup and spin the tube in a microcentrifuge at maximum speed for 30 seconds.
• Open the lid of the microcentrifuge tube and discard the microspin cup. The purified PCR product is in the bottom of the 1.5-ml microcentrifuge tube. Snap the lid of the microcentrifuge tube closed to store the purified PCR product.

HiFi assembly

• Setup the following reaction on Ice:

Note: Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of each insert. Use 5-fold molar excess of any insert(s) less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%. To achieve optimal assembly efficiency, design 15-20 bp overlap regions between each fragment.

• Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20°C for subsequent transformation.

  Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).

Heat Shock Transformation in E. coli

Overview:

The purpose of this protocol is to transform E. Coli to take up our plasmid DNA.

Materials:

• Frozen competent cells stored at -80C (25ul)
• Plasmid DNA
• Ice bucket
• 42C water bath
• SOC media
• Selection Plates
• Incubators
• Microfuge tubes

Procedure:

• Place a tube of 25 μL of cells on ice until thawed.
• Add 2 microliters of plasmid. Flick to tube 4-5 times to mix without vortexing.
• Place the mixture on ice for 30 minutes without mixing.
• Heat shock the mixture at 42C for exactly 30 seconds for DH5a cells, and 45 seconds for MC1061 cells.
• Place back on ice for 5 minutes. Do not mix.
• Add 475 μL of room temperature SOC to the mixture.
• Shake at 37 C for 120 minutes at 250rpm
• Warm selection plates to 37 C.
• Spread 100 μL of each dilution onto a selection plate and incubate overnight or for multiple days at 37 C.

Colony PCR

Materials:

• 10uM primer VF2
• 10uM primer VR
• 2X Q5 HotStart Master Mix
• Distilled water
• Agar plate with bacterial colonies
• PCR tubes
• Toothpicks
• Collecting Bacterial Cultures + Reaction Setup:

Be sure that you save each bacterial colony so that if the PCR shows what you want, you can go back to the original bacterial cells. You can do this by dipping the toothpick in step 5 below into liquid media or using to swab an agar plate before adding the DNA into the PCR tube.

• Take a toothpick and mark out 10 colonies that you want to analyze.
• Lightly touch the toothpick onto the colonies, dip into 100uL of water.
• Use 1uL of that water/cell concentration for template DNA for the rest of the PCR reaction.
• PCR reaction:
  • Used 2x PCRBIO HS Taq Mix Red
  • 12.5uL of 2x Taq
  • 1uL of Forward primer
  • 1uL of Reverse primer
  • 1uL of template DNA
  • 9.5uL of deionized water
• Run samples in thermocycler: 35 cycles at 56°C annealing temperature, 2 minutes for extension time

SM17 Medium Preparation

Overview:

This protocol was used to prepare SM17 media, a version of M17 media that was supplemented with Sucrose for optimal growth of L. Lactis, acting as a carbon source for the bacteria. This protocol makes 1L of media.

Materials:

• M17 Broth Powders
• Sucrose
• Water
• Plates
• Bottles
• Agar
• Autoclave Tape

Procedure:

• Suspend 42.25 grams of M17 broth powder in 1000 ml of purified/distilled water.
• 171g of Sucrose was added to bring the solution to 0.5M.
  • Optional: If preparing plates, 15g of Agar could be added to make 1L worth of plates. If making plates, 20 ml of media should be used for each, totalling 50 plates per liter of media.
• The solution was separated into bottles of equal volume.
• Autoclave tape was added to all the bottles.
• The media was autoclaved at 15 lb of pressure (121 celsius) for 15 minutes.

Plasmid Prep

Overview: The objective of miniprep was to extract plasmid DNA from E. coli.

Materials/Reagents:

• All those are included in the kit.

Procedure:

• Pellet 1ml of cells.
• Resuspend the pelleted cells in 250 µL of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
  • Note. Ensure RNase A has been added to the Resuspension Solution
• Add 250 µL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
  • Note. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
• Add 350 µL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.
  • Note. It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should become cloudy.
• Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
• Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
  • Note. Close the bag with GeneJET Spin Columns tightly after each use!
• Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
  • Note. Do not add bleach to the flow-through.
• (For EndA+ strains which have high level of nuclease activity only - optional): Wash the GeneJET spin column by adding 500 µL of Wash Solution I (#R1611, diluted with isopropanol) and centrifuge for 30-60 sec. Discard the flow-through.
  • Note. This step is essential to remove trace nuclease activity.
• Add 500 µL of the Wash Solution (diluted with ethanol prior to first use as described on p.3) to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
• Repeat the wash procedure (step 8) using 500 µL of the Wash Solution.
• Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
• Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 µL of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.
  • Note. An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%. For elution of plasmids or cosmids >20 kb, prewarm the Elution Buffer to 70°C before applying it to silica membrane.
• Discard the column and store the purified plasmid DNA at -20°C.

Lactococcus Lactis electroporation and competent cell prep

Using Rice IGEM 2018 Protocol

Making electrocompetent cells:

• Grow cells overnight at 30 °C to an O.D620 of 0.7.
• Wash twice with ice-cold washing solution.
• Resuspend cells in 1/100 volume of electroporation solution.
• Keep on ice.

Electroporation of cells:

• Add 0.25 µg plasmid DNA (in water) to 100 µl of electrocompetent cells.
• Homogenize by gently mixing with pipette several times.
• Transfer mixture into cuvette.
• Wipe moisture from the cuvette and insert the cuvette into the device.
• Electroporation:

Mode	Prokaryotes
Voltage (V)	2000 volts
Time Constant (T)	5 minutes

• Add 1 ml ice-cold complex medium, incubate 2 hours at 30 °C.
• Plate diluted cells on selective chloramphenicol plates.
• Incubate 2 days at 30 °C.

Expected results:
Transformation efficiency up to 1.4 x 106 transformants/µg of DNA.