Team:Thessaloniki/Protocols

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Competent cells preparation

Introduction

Before startting the procedure be sure to have the following autoclaved:

  1. 2 x 1 L erlenmeier flask
  2. 100 mL erlenmeier flask
  3. 2 x 500 mL centrifuge bottles
  4. 350 mL 0.1 M CaCl2
  5. 100% Glycerol

Materials

  1. LB Agar (500mL - Autoclaved): 7 g Agar, 10 g LB Broth, 15 mL NaOH Fill with ddH2O to 500 mL
  2. LB Medium (500 mL - Autoclaved): 10 g LB Broth, 150 μL NaOH Fill with ddH2O to 500 mL
  3. Cells

Procedure

Plating:

  1. Plate the cells on a plate. If the cells have any drug resistance integrated in them, use LB-Agar plates containing this resistance; otherwise use plain LB-Agar plates.

Growing:

  1. Pick one colony and grow overnight in 25 mL LB at 37°C (containig the drug if resistance is present).
  2. Transfer the cells to 500 mL LB final volume.
  3. Incubate at 37°C for ~2.5 hours (until OD reaches ~0.4).
  4. Centrifuge cells at 4°C for 15 minutes at 5000 RPM (Sorval).
  5. Discard the supernatant and resuspend the pellet in 250mL 0.1M CaCl2.
  6. Incubate on ice for 20 minutes.
  7. Centrifuge again as before (at 4°C for 15 minutes at 5000 RPM (Sorval)).
  8. Discard the supernatant and resuspend the pellet in 43 mL 0.1 M CaCl2.
  9. Incubate for 2-12 hours (usually 4 hours).

Storing

  1. Add 7 mL 100% Glycerol and mix well.
  2. Distribute in 1.5 mL tubes.
  3. Quick freeze in liquid nitrogen.
  4. Store at -80°C.
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Beta-galactocidase Assay

In short, the protocol consists of measuring the cell density of a culture of bacteria (Abs600), then removing an aliquot of the cells from the cuvette and mixing them with a "permeabilization" solution that contains detergent which disrupts the cell membranes (but leaves the β-Gal intact). This kills the cells and stops translation. After incubation, an ONPG "substrate" solution is added and the yellow color allowed to develop. A "stop" solution is then added and the absorbance of o-nitrophenol is measured.

  1. Grow cultures under the desired contitions.
  2. Pre-measure 80 μL aliquots of permeabilization solution into 1.5 mL microfuge tubes.
  3. Measure and record Abs600.
  4. Remove a 20 μL aliquot of the culture from the cuvette and add it to the 80 μL of permeabilization solution.
The sample is now stable for several hours.
  1. After the last sample is taken, move the samples and the Substrate solution to the 30 °C warm room for 20-30 minutes.
  2. Add 600 μL of Substrate solution to each tube and note the time of addition.
  3. After sufficient color has developed, add 700 μL of Stop solution, mix well, and note the stop time.
  4. After stopping the last sample (approximately about 30-90 minutes), transfer the tubes to a microfuge and spin for 5-10 minutes at full speed.
  5. Carefully remove the tubes from the centrifuge and measure the absorbance at Abs420 by transfering solution from the top of the tubes to cuvettes. The estimated absorbance is measured between 0.05 and 1. However, tis range is not restrictive.
Calculate Miller Units as:

1000* (Abs420) / ((Abs600 of culture sampled)*(volume[0.02mL])*(reaction time))

Permeabilization Solution production:

Each sample contains 80 μl.

Permeabilization Solution includes:

100 mM dibasic sodium phosphate (Na2HPO4)
20 mM KCl
2 mM MgSO4
0.8 mg/mL CTAB (hexadecyltrimethylammonium bromide)
0.4 mg/mL sodium deoxycholate
5.4 μL/mL beta-mercaptoethanol

Substrate solution production:

Each sample contains 600 μl.

Substrate Solution includes:

60 mM Na2HPO4
40 mM NaH2PO4
1 mg/mL o-nitrophenyl-β-D-Galactoside (ONPG)
2.7 μL/mL β-mercaptoethanol

Stop solution production:

Each sample contains 700 μl.

1 M Sodium Carbonate (Na2CO3)

The high pH of the stop solution denatures the β-Gal and approximately doubles the yellow color of the reaction.

Nds DNA Gates Cloning and Preparation

Introduction

The cloning process of the Nds DNA Gates described by D. Soloveichik et al. (Nature Nanotechnology, 2013) and Yuan-Jyue Chen et al. (Journal of Visualized Experiments, 2015) into bacterial cells and the subsequent isolation of the Gates through enzymatic digestion are described in this protocol.

Procedure

Day 1: Cloning of Nds DNA Gates into plasmids - Golden Gate Assembly

  1. After receiving the ordered DNA, spin the tubes containing genetic blocks at 10,000-14,000 g for 1 minute to ensure that all dried DNA is at the bottom of the tube.
  2. Resuspend the dried genomic blocks in DNAse-free water to achieve a final concentration of 10 ng/μL. NOTE: Alternatively, DNA can be resuspended using 1x Tris ethylenediaminetetraacetic acid (EDTA) buffer ( 1x TE Buffer: 10 mM Tris and 1 mM EDTA, pH 8.0). However, EDTA is a chelating agent for divalent cations and could inhibit PCR.
  3. Insert the BsaI restriction sites in the Promoter-RBS-AmilCP-PSB1C3 ligated plasmid by PCR, using Damalas1 and Damalas2 primers.
  4. Digest Promoter-RBS-AmilCP-PSB1C3 ligated plasmid with BsaI. Set the restriction digestion reaction as follows:

    For 20 μL reaction, add in the following order:
    ddH2O (to 20 μL reaction volume)
    DNA (the desired volume)
    2 μL 10x CutSmart Buffer
    0.5 μL BsaI-HFv2

  5. Incubate the reaction mix at 37oC for 1 hour.
  6. Heat the reaction mix to 80oC for 20 minutes to inactivate BsaI-HFv2.
  7. Run a 0.8% agarose gel electrophoresis to separate the PSB1C3 plasmid backbone (See Agarose Gel Electrophoresis Protocol).
  8. Gel purify BsaI-digested PSB1C3 plasmid backbone using NEB Monarch DNA Gel Extraction Kit, following the manufacturer's instructions.
  9. Measure the concentration of gel-extracted PSB1C3 plasmid backbone using a NanoDrop device.
  10. Ligate PSB1C3 plasmid backbone with the inserts (Input, Join and Fork Gates) according to the instructions provided by NEB's Golden Gate Assembly Protocol for Using NEB Golden Gate Assembly Kit (BsaI-HFv2)(E1601). Set the ligation reaction as follows:

    For 50 μL reaction, add in the following order:
    ddH2O (to 50 μL reaction volume)
    DNA (50 ng for PSB1C3 plasmid backbone, 2:1 insert:vector molar ratio).
    5 μL T4 DNA Ligase Buffer
    1 μL NEB Golden Gate Assembly Mix

  11. Incubate the reaction mix at 37oC for 5 minutes.
  12. Heat the reaction mix at 60oC for 5 minutes in order to inactivate the enzyme.
  13. Transform the Golden Gate Assembly products into E.coli competent cells and plate on LB agar plates containing chloramphenicol antibiotics at a working concentration of 34 μg/mL (prepared by adding 50 μL of 34 mg/mL chloramphenicol solution to a Falcon tube containing 50 mL of liquified LB agar).

Day 2: Bacterial culture amplification

There are two options: Culture for mini- and culture for midi-prep. The second option is strongly recommended for maximum plasmid DNA yield.

Option 1: Preparation of culture for mini-prep

  1. Add 3 mL of enriched medium (LB Medium or Terrific Broth) in a sterile tube.
  2. In the same tube, add 3 μL of 34 mg/mL chloramphenicol, in order to achieve the same working concentration of antibiotic (34 μg/mL) with the chloramphenicol selective plate.
  3. The colonies containing the assembled plasmid should be white in color.
  4. With the help of a pipette tip, pick a single colony from the chloramphenicol selective plate and add it in the tube. Incubate the culture at 37oC overnight with vigorous shaking (200-300 rpm). Typically, incubate for 16-24 hours. Optimally, 16-18 hours.

Option 2 (recommended): Preparation of culture for midi-prep

  1. Add 100 mL of enriched medium (LB Medium or Terrific Broth) in a sterile conical flask.
  2. In the same conical flask, add 100 μL of 34 mg/mL chloramphenicol, in order to achieve the same working concentration of antibiotic (34 μg/mL) with the chloramphenicol selective plate.
  3. The colonies containing the assembled plasmid should be white in color.
  4. With the help of a pipette tip, pick a single colony from the chloramphenicol selective plate and add it in the tube.
  5. Incubate the culture at 37oC overnight with vigorous shaking (200-300 rpm). Typically, incubate for 16-24 hours. Optimally, 16-18 hours.

Day 3: Plasmid extraction and enzymatic processing

  1. For Option 1: Extract the plasmid DNA from the bacterial culture using NEB Monarch Plasmid Miniprep Kit, following the manufacturer's instructions. For Option 2: Extract the plasmid DNA from the bacterial culture using Macherey-Nagel NucleoBond Xtra Midi kit, following the manufacturer’s instructions.
  2. Measure the purified plasmid DNA concentration using a NanoDrop device.
  3. Digest the purified plasmid DNA with restriction enzyme PvuII for 1 hour at 37oC to cut the DNA Gates from the plasmid. Typically digest the plasmid with 4 units of PvuII per 1 μg of plasmid. Set the restriction digestion reaction as follows:

    For example, for a 50 μL reaction using 1 μg DNA, add in the following order:

    ddH2O (to 50 μL reaction volume)
    1 μg DNA (volume depends on DNA concentration)
    5 μL 10x NEBuffer 3.1
    0.4 μL Pvull

    Note that the entire quantity of midi-prepped DNA can be used. In this case, calculate this quantity by multiplying concentration by the sample volume and use 0.4 μL of PvuII (containing 4 units) per 1 μg of DNA.

    WARNING: The addition of excess amounts of enzymes may lead to high amounts of initial circuit leakage, which is most likely caused by over-digestion. This issue can be addressed by optimizing the enzyme amounts as above.
  4. Pvull is not heat-inactivatable. Therefore, ethanol precipitation of the samples must be performed. Add 2 volumes of ice-cold absolute ethanol to each sample.
  5. Incubate the mixture at -80oC for at least 1 hour (this mixture can also sit at -80oC for overnight).
  6. Centrifuge at 10,000-14,000 g at 0oC for 30 minutes.
  7. Carefully remove the supernatant using a pipette.
  8. Add 1,000 μL (1 mL) of room-temperature 95% ethanol to the sample and invert 10-15 times.
  9. Centrifuge at 10,000-14,000 g at 4oC for 10 minutes.
  10. Carefully remove the supernatant using a pipette and air dry on bench until ethanol has completely evaporated.

    WARNING: Ethanol in the samples may inhibit enzymatic, as well as strand displacement reactions. Therefore, there must be no ethanol present in the samples before resuspension.
  11. Resuspend the DNA pellets in an appropriate volume of nuclease-free H2O (typically 50-100 μL).
  12. Measure the concentration of resuspended DNA using a NanoDrop device.

    NOTE: The NanoDrop device measures the overall concentration of DNA in the sample, both the cut Gates and the plasmid backbone. This is not a problem, as the amount of nicking enzyme used during the nicking digestion in the next step is calculated per certain amount of overall plasmid-derived DNA, including both cut Gate and backbone. Do not gel extract the PvuII-digested DNA fragments prior to the nicking restriction, because gel extraction dramatically reduces the concentration of isolated sample DNA, making it impossible to add the right amount of nicking enzyme that corresponds to such a small amount of DNA during the next step.


  13. Digest Join Gates with nicking enzyme Nb.BsrDI at 65oC for 1 hour using 4 units of enzyme per 1 μg of plasmid-derived DNA. Set the nicking digestion reaction as follows:

    For example, for a 50 μL reaction using 1 μg DNA, add in the following order:

    ddH2O (to 50 μL reaction volume)
    1 μg DNA (volume depends on DNA concentration)
    5 μL 10x CutSmart Buffer
    0.4 μL Nb.BsrDI

    Note that the entire quantity of PvuII-digested and ethanol precipitated DNA can be used. In this case, calculate this quantity by multiplying concentration by the sample volume and use 0.4 μL of Nb.BsrDI (containing 4 units) per 1 μg of DNA.

    WARNING: The addition of excess amounts of enzymes may lead to high amounts of initial circuit leakage, which is most likely caused by over-digestion. This issue can be addressed by optimizing the enzyme amounts as above.


  14. Digest Input Gates and Fork Gates with nicking enzyme Nt.BstNBI at 55oC for 1 hour using 8 units of enzyme per 1 μg of plasmid-derived DNA. Set the nicking digestion reaction as follows:

    For example, for a 50 μL reaction using 1 μg DNA, add in the following order:

    ddH2O (to 50 μL reaction volume)
    1 μg DNA (volume depends on DNA concentration)
    5 μL 10x NEBuffer 3.1
    0.8 μL Nt.BstNBI

    Note that the entire quantity of PvuII-digested and ethanol precipitated DNA can be used. In this case, calculate this quantity by multiplying concentration by the sample volume and use 0.8 μL of Nt.BstNBI (containing 8 units) per 1 μg of DNA.

    WARNING: The addition of excess amounts of enzymes may lead to high amounts of initial circuit leakage, which is most likely caused by over-digestion. This issue can be addressed by optimizing the enzyme amounts as above.

  15. Inactivate Nb.BsrDI by heating the sample containing Join Gates at 80oC for 20 minutes. Inactivate Nt.BstNBI by heating the sample containing Input or Fork Gates at 80oC for 20 minutes.
  16. Enzymes and enzyme buffers used should not be present in the samples used in the kinetic experiments, due to changing the optimized strand displacement reaction conditions. Therefore, ethanol precipitation of the nicked gates must be performed. Add 2 volumes of ice-cold absolute ethanol to each sample.
  17. Incubate the mixture at -80oC for at least 1 hour (the mixture can also sit at -80oC for overnight).
  18. Centrifuge at 10,000-14,000 g at 0oC for 30 minutes.
  19. Carefully remove the supernatant using a pipette.
  20. Add 1,000 μL (1 mL) of room-temperature 95% ethanol to the sample and invert 10-15 times.
  21. Centrifuge at 10,000-14,000 g at 4oC for 10 minutes.
  22. Carefully remove the supernatant using a pipette and air dry on bench until ethanol has completely evaporated.

    WARNING: Ethanol in the samples may inhibit enzymatic, as well as strand displacement reactions. Therefore, there must be no ethanol present in the samples before resuspension.

S.O.C. Medium Production

  1. Add 20 g Tryptone, 5 g Yeast extract and 0.5 g CaCl2 in a falcon tube and shake until dissolved.
  2. Add 10 mL of 250 mM KCl (1.86 g KCl in 100 mL ddH2O).
  3. Adjust the pH to 7.0 with 5 N NaOH (~0.2mL).
  4. Adjust the volume of the solution to 1 L with ddH2O.
  5. Sterilize by autoclaving.
  6. Add 5mL of a sterile 2 M MgCl2 solution (19g MgCl2 in 90mL ddH2O. Add ddH2O until the volume reaches 100 mL and autoclave).

After S.O.B. Medium reaches 60°C or less add 20 mL of 1 M Glucose (18 g glucose in 90 mL ddH2O. After dissolved, adjust the volume to 100 mL with ddH2O and sterilize by filtering).

Terrific Broth Production

Quantity

47.6 g per liter

Preparation Note

Supplement: 8 mL glycerol per liter

Reconstitution

Preparation instructions

  1. Suspend 47.6 g and 8 mL glycerol in 1 L of distilled water.
  2. Autoclave for 15 minutes at 121 °C.

Parts’ Cloning

Once the DNA is resuspended in any of the wells of the iGEM Distribution kit, the parts were transformed in DH5 alpha cells, according to the following protocol.

a.Transformation

Introduction

The process of inserting plasmid DNA into chemically competent cells.

Materials

  • Chemically competent E.coli cells
  • DNA to be transformed
  • LB agar plates with appropriate antibiotic for selection
  • Ice

Method

  1. Thaw 50 μL of competent cells on ice.
  2. Add DNA (For transformation of ligated products add 5 μL of the ligation reaction mix, whereas for transformation of known plasmids add approximately 10-100 μL of DNA.)
  3. Incubate cells on ice for 30 minutes.
  4. Heat shock cells at 42oC for 45 seconds.
  5. Incubate on ice for 5 minutes.
  6. Add 1mL S.O.C. or LB Broth.
  7. Incubate cells for 1 hour at 37oC in a shaking incubator (200-250 rpm)
  8. Centrifuge at 8000 rpm for 3 minutes.
  9. Remove 850 μL of supernatant.
  10. Resuspend pelleted cells in the remaining LB Broth or S.O.C.
  11. Plate resuspended cells onto LB agar plates containing the appropriate antibiotic.
  12. Let the plates dry inside the incubator for approximately 30 minutes.
  13. Flip the plates upside down and incubate for 16-18 hours.

After a colony PCR conducted following the protocol described below and the manufacturer's instructions of the Phusion High Fidelity PCR Master Mix with HF Buffer from New England Biolabs, the right colonies were picked and incubated in a shaking incubator for 16-18 hours.

b.Colony PCR protocol

Introduction

Colony PCR is a convenient high-throughput method for determining the presence or absence of insert DNA in plasmid constructs. The following protocol is proposed by Promega Corporation.

Materials and instruments

  • GoTaq G2 Flexi DNA Polymerase (5 units/μL)
  • 5x Green or Colorless GoTaq Flexi Buffer
  • MgCl2 Solution 25 mM
  • dNTPs 10 mM each
  • Upstream and downstream primers
  • Template DNA
  • Nuclease-free ddH2O
  • Thermal cycler
  • PCR tubes
  • Eppendorf 1.5 mL tubes

Preparation of template DNA

  1. Circle a certain number of colonies from a plate. Make sure the colonies are quite large, so as that not the entire colony is used for Colony PCR, but a part of it. There should be a sufficient quantity of cells for subsequent picking, in case the Colony PCR reveals that colony cells have been transformed with the right plasmid construct.
  2. Get as many sterilized Eppendorf 1.5 mL tubes as the number of colonies selected.
  3. Add 15 μL of nuclease-free ddH2O in each Eppendorf tube.
  4. Carefully pick a part of each colony using a sterilized pipette tip. Touch the tip on a sterile LB agar plate with the same antibiotic as the plate from where the colony was selected, so as to culture independently the selected colony's cells. Throw the tip inside one of the Eppendorf tubes.
  5. Leave the Eppendorf tubes at room temperature for 10 minutes, so as that the cells are resuspended in the amount of ddH2O added in the tubes.
  6. Lyse the resuspended cells by heating the Eppendorf tubes at 80oC for 20 minutes.

Preparation of Master Mix

  1. The mix should be adequate for a number of reacrions equal to the number of colonies selected from all plates plus a negative and a positive control, plus 1 (e.g. for 3 selected colonies, a Master Mix for 3 (colonies) + 1 (negative control) + 1 (positive control) +1 = a Master Mix for 6 reactions should be prepared).
  2. For each reaction, the following are added:
    8.875 μL Nuclease-free ddH2O
    5 μL 5x Green or Colorless GoTaq Flexi Buffer (for a final concentration of 1x)
    3 μL MgCl2 Solution 25 mM (for a final concentration of 3 mM)
    0.5 μL dATPs 10 mM (for a final concentration of 0.2 mM)
    0.5 μL dTTPs 10 mM (for a final concentration of 0.2 mM)
    0.5 μL dCTPs 10 mM (for a final concentration of 0.2 mM)
    0.5 μL dGTPs 10 mM (for a final concentration of 0.2 mM)
    0.5 μL upstream primer 10 mM (for a final concentration of 0.2 mM)
    0.5 μL downstream primer 10 mM (for a final concentration of 0.2 mM)

Preparation of Reaction Mix

  1. Get as many PCR tubes as the number of colonies selected plus one for negative control sample plus one for positive control sample.
  2. Add 19.875 μL of Master Mix in each PCR tube.
  3. For each colony, add 5 μL of the lysate in the respective PCR tube. The lysate contains the template DNA to be amplified.
  4. For negative control sample, add 5 μL nuclease-free ddH2O in the respective PCR tube.
  5. For positive control sample, in the respective PCR tube add 4.7 μL nuclease-free ddH2O and 0.3 μL of a template DNA sample of verified identity, which can be amplified by the use of the same primers as the DNA from the colonies.
  6. Finally, add 0.125 μL of GoTaq Flexi DNA Polymerase in each sample. GoTaq Flexi DNA Polymerase should be the last component to be added to each reaction mix.

PCR Program

  1. Denaturation: Following an initial 2-minute 94-95oC denaturation, denaturation steps should be between 15 seconds and 1 minute per cycle.
  2. Annealing: Optimize the annealing conditions by performing the reaction with an annealing temperature approximately 5oC below the calculated melting temperature of the primers and increasing the temperature in increments of 1oC. The annealing step is typically 15 seconds to 1 minute.
  3. Extension: The extension reaction is typically performed at the optimal temperature for Taq DNA polymerase, which is 72-74oC. Allow approximately 1 minute for every 1 kb of DNA to be amplified. A final extension of 5 minutes at 72-74oC is recommended.
  4. Soak: If the thermal cycler has a refrigeration or "soak" cycle, the thermal cycler can be programmed to hold the tubes at 4oC for several hours after amplification. This cycle minimizes polymerase activity, which might occur at higher temperatures, although this is not usually a problem.
  5. Cycle Number: Generally, 25-30 cycles result in optimal amplification of desired products. Up to 40 cycles may be performed, especially to detect low-copy targets.

c.Agarose Gel Electrophoresis

Introduction

We use this type of electrophoresis when separating and purifying DNA fragments derived from mini-prepped plasmids digested with restriction endonucleases, PCR or Colony PCR products. It is used in general for separation and purification of DNA fragments larger than 100 bp. For purification of oligonucleotides, PAGE electrophoresis is preferred (see PAGE Purification).

Materials

  • Agarose Gel Electrophoresis device
  • Agarose
  • Ethidium Bromide stain (1% in DMSO)
  • NEB Gel Loading Dye Purple (6x)
  • DNA Ladder (100 bp)
  • DNA samples
  • 1x TAE Buffer

Preparation of 1.0% agarose gel

  1. Agarose w/v% percentage of the gel may vary depending on the purpose of the electrophoresis analysis (separation, extraction etc.), the size of the DNA fragments analyzed and the voltage applied to the gel. For gel extraction, gels of 0.8-1.0% agarose are preferred, while a bit higher percentages of agarose can be used such as 1.5% or 2.0%, for better separation when analysing DNA fragments for diagnostic purposes (for Colony PCR products, diagnostic restriction endonuclease digestion etc.).
  2. Add 70 mL of 1x TAE Buffer in a conical flask of 150 mL.

    WARNING: we use the specific conical flask which is used only for this procedure, due to the fact ethidium bromide, a carcinogen, is added to the same flask during the next steps of the present protocol. Due to the addition of ethidium bromide, this flask must not be used for any other experimental procedure and must be stored in a place separate from other glassware. Other flasks should also not be used for this procedure, so as not to be contaminated with ethidium bromide.

  3. For a 1% agarose gel, add 0.7 g in the same conical flask.
    NOTE: It is important to add 1x TAE Buffer first and then agarose, unlike in other procedures where the solid is subsequently dissolved with the addition of solvent.
  4. Agarose is not soluble in water at room temperature. Therefore, the conical flask containing the agarose-TAE mixture is heated in a microwave oven for a few seconds and shaken over brief periods of time. When agarose is completely dissolved, heating is stopped.
  5. The flask is let to cool off a bit before the addition of ethidium bromide.
    NOTE: Ethidium bromide is degraded at high temperatures.
  6. In the same conical flask, 3 μL of ethidium bromide are added and mixed uniformly with the agarose-water mixture.
  7. Turn the cassette of the electrophoresis device, such as the red lines are oriented towards the sides of the device, so that the gel mixture does not leak from the cassette to the body of the device.
  8. The mixture of the conical flask is added to the cassette of the electrophoresis device.
  9. Immediately insert the appropriate comb at the top side of the device ( the side where the (-) cable is plugged). For gel extraction the 10-tooth comb is preferred to other combs with more teeth, because it leaves larger wells, which can be filled with larger quantity of sample to be extracted.
  10. After about 20 minutes the gel should have been formed.

Sample loading

  1. After formation of the gel, remove the comb carefully, so that the sample loading wells are revealed.
  2. Turn the cassette of the electrophoresis device, such as the red lines are oriented towards the two edges of the electrophoresis device.
  3. Add the running buffer (1x TAE) in the electrophoresis device, until the surface of the liquid reaches the level of the line indicated on the side of the device. The gel should be completely covered by a short layer of running buffer.
  4. Mix the 20 μL volume of the sample with 4 μL Loading Dye or the 25 μL volume of the sample with 5 μL Loading Dye.
  5. Load 4 μL of the 100 bp DNA Ladder into a well.
  6. Plug the device with the power supply and run the electrophoresis at 70 V for about 2 hours (until the samples have moved to the middle of the gel or a little bit further).

Band visualization

  1. After the end of the electrophoresis, unplug the device and move the cassette to a UV illuminator. With the help of the DNA Ladder correspond each band to a DNA fragment of specific size.
  2. If gel extraction of DNA fragments is required, cut the bands which contain the desired DNA fragments to be extracted, wearing a UV filter mask.
  3. Add each cut band in a 1.5 mL Eppendorf tube.

d.Colony Picking and Culture

Introduction

This is the procedure followed for the selective growth of a colony that contains the correct plasmid construct, in order to amplify the plasmid DNA so as to subsequently isolate it at a large quantity.

Materials

  • DH5 alpha or XL-1 competent bacteria with the desired plasmid DNA construct
  • Terrific Broth medium
  • Chloramphenicol 34 mg/mL in ethanol (for colonies containing the PSB1C3 plasmid)
  • Sterile tubes
  • Pipette tips

Culture medium preparation

  1. For a culture to be mini-prepped, add 5 mL of Terrific Broth medium in a sterile tube. For a culture to be midi-prepped, add 100-150 mL of Terrific Broth medium in a sterilized conical flask. Make sure that enough quantity of air is left inside the tube or flask, because sufficient air is vital for the normal growth of the cultured bacteria.
  2. For colonies containing the PSB1C3 plasmid, add 1 μL of antibiotic per 1 mL of culture medium. For colonies with the PSB1C3 plasmid, the antibiotic is chloramphenicol with initial stock concentration of 34 mg/mL, which, after the addition in the culture medium, yields a final working concentration of 34 μg/mL.

Colony selection and picking

  1. Mark the desired number of colonies from an LB agar plate. If the plate has been streaked with bacteria transformed with a ligated plasmid construct, make sure that the selected colony has been confirmed to contain the correctly assembled construct by Colony PCR and subsequent Agarose Gel Electrophoresis of the Colony PCR product on a 2% agarose gel.
  2. Pick each colony with the help of a sterile pipette tip handled with a pair of tongs. Make sure that the pipette tip reaches the bottom of the plate, so as that the entire mass of the colony is being lifted.
  3. Drop the pipette tip inside the sterile tube or the flask containing the antibiotic-enriched culture medium.
  4. Incubate the tube or flask containing the culture at 37oC for 16-18 hours (16 hours is the optimal amount of time for bacterial culture growth).

e.i.Mini-prep of plasmid DNA from bacterial culture

The isolation of plasmid DNA from small-scale bacterial cultures (mini-prep) is performed using NEB Monarch Plasmid Miniprep Kit, according to the manufacturer’s instructions.

e.ii.Midi-prep of plasmid DNA from bacterial culture

The isolation of plasmid DNA from middle-scale bacterial cultures (midi-prep) is performed using Macherey-Nagel NucleoBond Xtra Midi kit, according to the manufacturer’s instructions.

f.Polymerase Chain Reaction (PCR)

Introduction

This protocol is proposed by New England BioLabs for in vitro amplification of small quantities of a certain DNA sequence, for the purpose of gaining greater quantities of DNA for subsequent cloning procedures, such as restriction enzyme digestion and ligation. After PCR, the PCR product should either run on a 0.8% Agarose Gel Electrophoresis and subsequently be extracted from the gel bands, or be purified from the PCR mixture by PCR Clean-up, in order to be ready to be used for subsequent cloning procedures, such as digestion with restriction endonucleases.

Materials

  • 2x Phusion High Fidelity PCR Master Mix
  • Upstream and downstream primers
  • DMSO
  • Template DNA
  • Nuclease-free ddH2O
  • Thermal cycler
  • PCR tubes
  • Eppendorf 1.5 mL tubes

Procedure

  1. Get as many PCR tubes as the number of samples plus one for negative control sample pus one for positive control sample (a sample of known consistency that has been previously successfully amplified).
  2. In each PCR tube add the following, in the following order:
    8.95 μL Nuclease-free ddH2O
    1.25 μL upstream primer 10 mM (for a final concentration of 0.5 mM)
    1.25 μL downstream primer 10 mM (for a final concentration of 0.5 mM)
    0.75 μL DMSO (for a final concentration of 3% v/v)
    0.3 μL Template DNA (except for the negative control sample, where 0.3 μL Nuclease-free ddH2O are added instead)
    12.5 μL 2x Phusion High Fidelity PCR Master Mix (for a final concentration of 0.5 units/ 25 μL Phusion DNA Polymerase, 1.5 mM MgCl2 and 200 μM of each dNTP)

PCR Program

  1. Denaturation: An initial denaturation of 30 seconds at 98oC is sufficient for most amplicons from pure DNA templates. Longer denaturation times can be used (up to 3 minutes) for templates that require it. During thermocycling, the denaturation step should be kept to a minimum. Typically, a 5-10 second denaturation at 98oC is recommended for most templates.
  2. Annealing: Annealing temperatures required for use with Phusion tend to be higher than with other PCR polymerases. The calculator mNEB T should be used to determine the annealing temperature when using Phusion. Typically, primers greater than 20 nucleotides in length anneal for 10-30 seconds at 3oC above the Tm of the lower Tm primer. If the primer length is less than 20 nucleotides, an annealing temperature equivalent to the Tm of the lower primer should be used. A temperature gradient can also be used to optimize the annealing temperature for each primer pair. For two-step cycling, the gradient can be set as high as the extension temperature. For high Tm pairs, two-step cycling without a separate annealing step can be used.
  3. Extension: The recommended extension temperature is 72oC. Extension times are dependent on amplicon length and complexity. Generally, an extension time of 15 seconds per kb is recommended.
  4. Cycle number: Generally, 25-35 cycles yields sufficient product.

g.DNA extraction from agarose gel

The extraction of DNA from agarose gel bands is performed using NEB Monarch Gel Extraction Kit, according to the manufacturer’s instructions.

h.PCR Clean-up

The cleaning-up process of PCR product DNA from the PCR mixture is performed using Macherey-Nagel’s NucleoSpin Plasmid kit, according to the manufacturer’s instructions.

i.Digestion with Restriction Endonucleases

Introduction

This protocol is used for optimization of restriction enzyme digestions of DNA parts that follow BioBrick RFC[10] Standard Assembly, as described by New England BioLabs.

Materials

  • EcoRI 0.5 μL and SpeI 0.5 μL or XbaI 0.5 μL and PstI 0.5 μL or EcoRI 0.5 μL and PstI 0.5 μL
  • NEBuffer 2.1 10x 2 μL
  • DNA sample to be digested
  • ddH2O as much as it is required so as that the final reaction volume is 20 μL

Preparation of digestion reaction mix

  1. In an Eppendorf tube of 1.5 mL add the amount of water that is needed so as that the final reaction volume is 20 μL.
  2. Add 200 ng of the sample DNA.
  3. Thaw NEBuffer 2.1 10x completely and vortex it vigorously so that it is completely homogenized. Add 2 μL of NEBuffer 2.1 10x.
  4. Add 0.5 μL EcoRI and 0.5 μL SpeI (for Insert #1) or 0.5 μL XbaI and 0.5 μL PstI (for Insert #2) or 0.5 μL EcoRI and 0.5 μL PstI (for plasmid backbone).

Incubation

Incubate the Eppendorf tube that contains the reaction mixture at 37oC for 1 hour.

Heat-kill

After completion of the digestion reaction, inactivate the restriction enzymes by heating the Eppendorf tube at 80oC for 20 minutes.

j.Ligation Protocol with T4 DNA Ligase

Introduction

This is the protocol followed for the ligation of DNA parts digested according to the BioBrick RFC [10] Standard Assembly, as described by New England BioLabs.

Materials

  • 10x T4 DNA Ligase Reaction Buffer
  • T4 DNA Ligase
  • Vector DNA
  • Insert DNA (Insert #1 and Insert #2)
  • Nuclease-free ddH2O

Setup of the ligation reaction

  1. In an Eppendorf tube of 1.5 mL add the amount of ddH2O that is needed so as that the final reaction volume is 20 μL.
  2. Add the vector and each of the inserts at a molar ratio of 3 insert: 1 vector. The mass of each insert is calculated according to the following formula:
    Required insert mass (g) = desired insert:vector molar ratio x mass of vector (g) x ratio of insert to vector lengths
    Use NEBioCalculator to calculate molar ratios.
  3. Thaw T4 DNA Ligase Buffer 10x completely and resuspended at room temperature, so as that it is completely homogenized. Add 2 μL of T4 DNA Ligase Buffer 10x.
  4. T4 DNA Ligase should be added last. Add 1 μL of T4 DNA Ligase.

Incubation

Incubate the Eppendorf tube that contains the reaction mixture at room temperature for 20 minutes.

Heat-kill

After completion of the ligation reaction, inactivate T4 DNA Ligase by heating the Eppendorf tube at 65oC for 20 minutes.

DNA Gates Preparation

( Resuspension - Annealing - PAGE purification - Destaining - Real Time PCR ) After the resuspension and the annealing of the DNA Oligos according to the Integrated DNA Technologies’ instructions, our DNA circuits were through a 10% native PAGE purification, were stained with SYBR Gold and then destained before being measured in a Real Time PCR according to the following protocols.

Resuspension

  1. After receiving the single stranded oligos from DNA manufacturer, spin the tubes containing DNA at 10,000-14,000 x g for 1 minute to ensure that all dried DNA is at the bottom of the tube.
  2. Resuspend the DNA using ddH2O to achieve a final concentration of 100 μM (see IDT Oligonucleotide Specification Sheets).

Annealing

  1. The synthetic DNA gates used in our kinetic experiments are formed through annealing after mixing complementary single strands for each complex (Input, Join, Fork Gate and Reporter Complex) with nominally correct stoichiometry.
  2. Mix the complementary single oligo strands that comprise each complex in a PCR tube, as follows:

    Input Gate

    Input Gate (bottom strand) at 100 μM 10 μL
    n at 100 μM 12 μL
    tbb at 100 μM 12 μL
    tdd at 100 μM 12 μL
    Final volume 46 μL
    Final complex concentration 21.74 μM

    Join Gate

    Join Gate (bottom strand) at 100 μM 10 μL
    atb at 100 μM 12 μL
    btr at 100 μM 12 μL
    rtq at 100 μM 12 μL
    Final volume 46 μL
    Final complex concentration 21.74 μM

    Fork Gate

    Fork Gate (bottom strand) at 100 μM 10 μL
    i at 100 μM 12 μL
    tcc at 100 μM 12 μL
    tbb at 100 μM 12 μL
    trr at 100 μM 12 μL
    Final volume 58 μL
    Final complex concentration 17.24 μM

    Reporter Complex

    Reporter Bottom F at 100 μM 10 μL
    Reporter Top Q at 100 μM 13 μL
    Final volume 23 μL
    Final complex concentration 43.48 μM

    Note that the short top strands of Input, Join and Fork Gates are added in 20% excess, in order to maximize the efficiency of their annealing with the complementary long bottom strands. Reporter complexes are annealed with a 30% excess of the top strand with the quencher, in order to ensure complete quenching of fluorescent bottom strands [1].

  3. Insert the PCR tubes in a thermal cycler device and heat at 95oC for 5 minutes.
  4. Slowly drop the temperature to 20oC, at a rate of 0.1oC per minute. The slow decrease of temperature ensures the correct formation of complexes.
  5. The samples can be stored for long term at 4oC.

10% native PAGE purification with 1.0 mm thickness gel

10x TAE/Mg2+ buffer preparation:

  1. In a volumetric flask add 4.84 g of Tris base, 0.372 g of EDTA disodium salt dihydrate and 2.68 g magnesium acetate tetrahydrate.
  2. Dissolve the above ingredients with 80 mL deionized water.
  3. Adjust pH to 8.0 by adding acetic acid solution.
  4. Acetic acid 1 M solution preparation: dilute 0.572 mL glacial (≥99%) acetic acid with deionized water to a final volume of 10 mL.
  5. Dilute the solution to 100 mL.

For preparing 1 x TAE/Mg2+ buffer, dilute the above to 10x the volume with ddH2O.

Acrylamide/bis-acrylamide preparation:

  1. In a volumetric flask add 38.67 g acrylamide and 1.33 g bis-acrylamide for acrylamide/bis-acrylamide 29:1 or 38.00 g acrylamide and 2.00 g bis-acrylamide for acrylamide/bis-acrylamide 19:1.
  2. Dissolve the above ingredients to 100 mL with ddH2O.

    3. 10% APS Preparation:

  3. In a volumetric flask add 10 g ammonium persulfate.
  4. Dissolve and dilute to 100 mL with ddH2O.
  5. Store the solution in the fridge (-4oC) in order to prevent degradation of APS.

Gel mixture preparation:


  1. In a conical flask mix 10 mL of 40% acrylamide/bis-acrylamide (19:1) with 4 mL of 10x TAE/Mg2+ .
  2. Dilute the mixture to 40 mL with ddH2O.
  3. Add 10 mL of the mixture into a Falcon tube. Store the rest of the mixture in the fridge (4oC) for further uses. The conical flask containing the stock mixture should be covered with Parafilm.
  4. In the Falcon tube containing 10 mL of the acrylamide/bis-acrylamide mixture add 75 μL of 10% ammonium persulfate and 7.5 μL of TEMED to help polymerization of acrylamide/bis-acrylamide.
  5. Wear gloves. Work quickly after addition of TEMED to complete the gel before the gel polymerizes. Quickly up-and-down the mixture with a 10 mL serological pipette. Add the mixture between the glasses of the cassette.
  6. Immediately insert the appropriate comb into the gel, being careful not to allow air bubbles to become trapped under the teeth. The tops of the teeth should be slightly higher than the top of the glass. Clamp the comb in place with bulldog paper clips.
  7. Allow the acrylamide/bis-acrylamide mixture to polymerize for 30 - 60 minutes at room temperature.

Sample loading:

  1. Mix the annealed gates to a final concentration of 1 μM with 10x TAE/Mg2+ , to a final concentration of 1x, and glycerol, to a final concentration of 15%, as follows:

    Input Gate

    Substance Volume added Final concentration
    Annealed Input Gate at 21.74 μM 46 μL 1 μM
    10x TAE/Mg2+ 100 μL 1x
    100% glycerol 150 μL 15%
    ddH2O 704 μL
    Final volume: 1,000 μL

    Join Gate

    Substance Volume added Final concentration
    Annealed Join Gate at 21.74 μM 46 μL 1 μM
    10x TAE/Mg2+ 100 μL 1x
    100% glycerol 150 μL 15%
    ddH2O 704 μL
    Final volume: 1,000 μL

    Fork Gate

    Substance Volume added Final concentration
    Annealed Join Gate at 17.24 μM 58 μL 1 μM
    10x TAE/Mg2+ 100 μL 1x
    100% glycerol 150 μL 15%
    ddH2O 692 μL
    Final volume: 1,000 μL
  2. Fill the electrophoresis device with the running buffer (1 x TAE/Mg2+ ), with an amount of running buffer covering the gel.
  3. Remove the comb carefully, so that the sample loading wells are revealed.
  4. Mix 4 μL of Ladder DNA with 11 μL of 10% glycerol and add the mixture into one well. Load the samples into the wells, at a volume of 15 μL. The pipette should be kept inclined and inserted in the gel carefully, without reaching the bottom so as not to destroy the lower layers of the gel.
  5. Load the samples into the wells, at a volume of 15 μL. The pipette should be kept inclined and inserted in the gel carefully, without reaching the bottom so as not to destroy the lower layers of the gel.

Running process:

  1. Plug the electrodes to the power supply. Be careful to plug the black-labelled electrode with the black cable and the red-labelled electrode with the red cable. Insert the cables into their respectively colored receptors in the power supply device.
  2. Purification gels run at 160 V for about 2 hours, cooled by placing ice packs next to the electrophoresis device.

Staining with SYBR Gold:

  1. Unplug the electrophoresis device and remove the running buffer.
  2. Carefully remove the glasses of the cassette and place the gel in a staining container such as a petri dish, the lid of a pipette tip box, or a polypropylane container.
  3. Add enough SYBR Gold solution to completely cover the gel. A 50 mL volume is generally sufficient for staining most standard minigels.
  4. Protect the staining solution from light by covering it with aluminum foil or by placing it in the dark.
  5. Prewashes of gels are not required, even for gels containing urea, formaldehyde or glyoxalated samples. Removal of the glyoxal is also not necessary.
  6. Agitate the gel gently at room temperature.
  7. The optimal staining time is typically 10-40 minutes, depending on the thickness of the gel and the percentage of polyacrylamide. The gel is usually stained for 20 minutes.
  8. The staining solution may be stored in the dark and can be reused 3-4 times, although best results are obtained from fresh staining solution.

Viewing the gel:

  1. Stained gels may be viewed with 300 nm UV or 254 nm epi- or transillumination.
  2. Stained gels may also be visualized and analyzed with laser scanners. Maximum visible light excitation is 495 nm.
  3. Gels may be photographed using Polaroid 667 black-and-white print film and a SYBR photographic filter (S7569). When using Polaroid film and this filter, we find that when exciting gels at 300 nm using the FOTO/UV 450 transilluminator, a 0.5-1 .0 second exposure with an f-stop of 5.6 is generally optimal. Optimal photographic conditions should be determined empirically for other light sources.
  4. With 254 nm epi-illuminator, exposures of about 1 minute may be required for maximal sensitivity when using Polaroid film and the SYBR filter.

Cut and Elution:

  1. Cut gel bands crushed into small pieces.
  2. Soak in 1x TAE/Mg2+ for 48 hours at 25 °C.
  3. Centrifuge at >16,000 g for 15 min.
  4. Retain the upper part of the liquid.

SYBR Gold Destaining:
The SYBR Gold stain can be efficiently removed from nucleic acids by simply precipitating the DNA or RNA with ethanol. More than 97% of the dye is removed by a single precipitation step. More than 99% of the dye is removed if ammonium acetate is used as the salt in the precipitation procedure.

  1. Add one of the following salts to the nucleic acid sample, to the indicated final concentration: 200 mM NaCl (prepared by mixing 0.584 g NaCl with ddH2O to a final volume of 50 mL in a Falcon tube), or 300 mM sodium acetate with pH=5.2 (prepared by mixing 1.231 g sodium acetate with ddH2O and adjusting pH to 5.2 by adding about 311 μL of glacial acetic acid, to a final volume of 50 mL in a Falcon tube) or 2.0 M ammonium acetate (prepared by mixing 7.708 g ammonium acetate with ddH2O to a final volume of 50 mL in a Falcon tube). Mix the nucleic acid sample with the salt solution gently. 2.0 M ammonium acetate is the most preferable of the three, followed by 300 mM sodium acetate and 200 mM NaCl.
  2. Add 2 volumes of ice-cold absolute (100%) ethanol and mix well. For a quick procedure, incubate the sample at 0oC (on ice) for 30 minutes. For increased yield, incubate the sample at ≤-20oC for ≥1 hour.
  3. Pellet nucleic acids by centrifuging for at least 15 minutes at 10,000-14,000 g at 0oC.
  4. Remove the supernatant and wash the pellet with ice-cold 70% ethanol (prepared by mixing 70 mL absolute ethanol with ddH2O to a final volume of 100 mL).
  5. Centrifuge again for at least 15 minutes at 10,000-14,000 g at 4oC to pellet nucleic acids.
  6. Measure the DNA concentration by using a NanoDrop device.

Fluorescence measurements

  1. Set the temperature controller to 25oC and wait for the temperature to stabilize. Using a temperature controller can reduce variablility in the signal that can result from temperature variation.
  2. Set proper parameters for kinetic measurements in the data acquisition software of the Real-Time PCR instrument.
    • Set the slit width to 2.73 nm for both excitation and emission monochromators.
    • Set the integration time to 10 seconds for every 60 second time-point. Set the total measurement time to 24 hours.
    • Set the excitation/emission wavelengths to match the fluorophore used in the experiment (FAM): 495 nm/ 520 nm
  3. Add nuclease-free ddH2O and 10x Transcription Factor Binding Buffer containing 125 mM Mg2+ to a final concentration of 1x. For a 40 μL reaction volume, add 4 μL of 10x Transcription Factor Binding Buffer.
  4. Add 1 μg poly(dT-dA) DNA to the final mixture, in order to reduce nonspecific interactions of protein to DNA, as well as nonspecific binding of target DNA to pipette tips.

Transcription Factor Binding Buffer Preparation

Introduction

This is the buffer proposed by Christopher B. Phelps et al. for in vitro binding of NF-κB to its target DNA site. We use the same buffer for the binding of ELK1 to its target site as well.

Materials

  • 50 mM NaCl
  • 12.5 mM MgCl2
  • 20 mM Tris
    • Adjust pH to 8.0 with 37% HCl
  • 1 mM Dithiothreitol (DTT)
  • 0.25 mg/mL Bovine Serum Albumin
  • 1 μg poly(dT-dA) DNA
  • ddH2O

Preparation of 10 mL 10x Stock Buffer

  1. Weigh and add the following in a 15 mL sterile Falcon tube:
    1. 0.292 g sodium chloride (NaCl)
    2. 0.254 g magnesium chloride hexahydrate (MgCl2⦁6H2O)
    3. 0.242 g Tris
    4. 0.025 g BSA
    5. ddH2O
  2. Adjust pH to 8.0 by adding a few droplets (about 20 μL) of 37% HCl.
  3. After having added all ingredients except for poly(dT-dA) DNA, add 100 μL of 1M DTT on ice (0oC).
  4. Store the 10x Stock Buffer at -20oC.

Use

When needing to use the buffer, defrost it and add a quantity equal to 1/10 of the desired final volume of transcription factor binding reaction to the mixture.