Team:DTU-Denmark/Experiments

Experiments

If you've ever participated in iGEM, then you know just how many hours we have spent in the lab. Even though we didn't quite install half a dozen beds in the break room like we wanted to, we still felt like sharing our experiences with all of you. Behold! Our protocols.

Protocols

Lab friendly downloads available for all protocols

This protocol is the adapted protocol from 223.

Materials

  • Fungal plates (either streaked or 3-point)
  • Drigalski spatula
  • 500 mL shake flasks
  • Counting chamber
  • Solutions (APB, ATP, PCT, Milli-Q, TM)
  • 30 °C incubator with shaking
  • Sterile tea spoon
  • Mira cloth in funnel (sterile)
  • Glucanex
  • Magnet stirrer
  • Magnets
  • 50 mL sterile falcon tubes
  • 0.45 µm filters
  • 50 mL syringe
  • Centrifuge for falcon tubes

Media

  • Aspergillus transformation buffer (ATB)
  • Aspergillus protoplastation buffer (APB)
  • 500 mL shake flasks
  • YPD media

Procedure

Initiation

  • Streak spore suspension of host strain on YPD plates supplemented with uridine (for this particular fungus) and let grow for a week (there should be black spores!)
  • All solutions should be sterile.

Day 1 (inoculation)

  • Add 95 mL of YPD media supplemented with uridine to a shake flask and transfer 5 mL of the YPD media to a plate with A. niger. Collect conidia and spores from plate by carefully scraping off the conidia using a drigalski (the spores are hydrophobic and would therefore rather just fly around than actually get into suspension so be careful not to make a mess here). This should give a concentration of around 108 spores/100 mL. It is a good idea to make more than one shake flask at a time.
  • Incubate shake flasks at 30 °C, 150 RMP for 48 h.

Day 3 (mycelial harvest)

  • Place the sterile funnel with a mira cloth in to a sterile blue cap bottle and transfer the contents of the shake flasks to the mira cloth (content should be brown and thick).
  • Wash the mycelia using Aspergillus protoplastation buffer (APB) to remove residual glucose from the mycelia (this can inhibit protoplastation). You need to use quite a bit of APB. squeeze out remaining liquid using a sterile spoon. Then, transfer the mycelium to falcon tubes ( ≈2 g per tube => 1 shake flask ≈ 2 falcon tubes).

Protoplastation

  • Add glucanex to APB to get a final concentration of 40 mg glucanex per mL APB and dissolve glucanex via gentle magnetic stirring and no heat.
  • Sterile filter 20 mL of APB+glucanex to each falcon tube using a 0.45 µm filter and a 50 mL syringe (there is a bit of resistance in the filter but that's ok).
  • Shake/incubate enzyme-mycelium mix at 30 °C, 150 RPM for 2-3 h.
  • From now on, whenever you pipette anything with the cells in it, cut of the edge of the pipette tip and CAREFULLY(!) pipette the cells. If you don't do this, they break as they don't have a cell wall to keep them stable.
  • Evaluate the number and quality of protoplasts in a microscope and discard a batch that is too diluted (i.e. < 105 protoplasts/mL). Approved protoplast solutions are then diluted by pouring APB up to 40 mL. and the tubess are balanced. Dilute Aspergillus transformation buffer (ATB) to 1/2x with sterile Milli-Q water and carefully place 5 mL of this on top of the APB, creating an overlay. Centrifuge samples on rotor settings rotor code Sla-600TC; time: 13 min; Speed: 3000g; Temperatire: 16 °C, Acc: 2, Brake: 2 (NB! due to slow acc and brake, this takes forever!)
  • In the interphase between the two liquids, a halo of white slurry consisting of concentrated protoplasts should be observed. If there is cell wall debris mixed in with the protoplasts, that's ok. They can still be used. Withdraw the protoplasts with a pipette and wash them in a new falcon tube. Add ATB up to 40 mL and pellet the protoplasts at 3000g for 13 min (acc. 2, brake 2). Discard supernatant by decanting.
  • Count protoplasts in microscope by diluting a small sample 1:100.
  • Resuspend protoplasts in 4 mL ATB to obtain concentrated solution.

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A quick overview of the two different transformation protocols

Materials

  • DNA
  • Protoplasts
  • Falcon tubes
  • PCT
  • TM molten agar
  • TM plates
  • ATB

Procedure

Regular protocol

  • Add at MOST 20 µL DNA ( or max 25% of protoplast volume) and 100 µL of protoplasts in a 50 mL falcon tube.
  • Incubate in the falcon tube at RT for at least 30 min.
  • Add 1mL of PCT.
  • Gently mix by gently swirling the tube in a circular motion – careful they are fragile. Do not vortex or pipette mix.
  • Incubate for 5 min at RT.
  • Add 3 mL of ATB.
  • Add 12 mL of molten (40-45 °C) TM agar.
  • Immediately pour mixture directly onto TM plates, and swirl to spread mixture evenly.
  • Let the plates settle for a few minutes and incubate at 37 °C for 4 days.

Quick protocol

  • Add at MOST 25 µL (1500-5000 ng) DNA ( or max 25% of protoplast volume) and 100 µL of protoplasts in a 2 mL Eppendorf tube. 1 µL (100 ng) pac6 as positive control.
  • Add 150 μL PCT with large-nozzle pipette tip.
  • Gently mix by swirling – careful the protoplasts are fragile.
  • Incubate 10-30 min at room temperature.
  • Add 250 μL ATB.
  • Distribute transformation mix on osmotic-stabilized selective media and let the agar absorb the mix before incubating

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Protocol for producing a glycerol stock of E. coli adapted by Jacob Mejlsted from Addgene's protocol.

Materials

Consumables

  • 2 mL screw top tube or cryovial

Chemicals

  • 50% glycerol
  • Cell culture

Procedure

Making the stock

  • After having cultivated a culture overnight, add 500 µL culture to 500 µL 50% glycerol in the selected tube
  • The stock can now be frozen at -80 °C
    If the stock is repeatedly thawed and frozen, it will reduce the shelf life of the culture

Using the stock

  • To recover bacteria, take a innoculation loop, toothpick or pipete tip and scrape a small amount of bacteria off the top.
  • Transfer this to a plate or a tube with liquid media for growth.
    Normally, media with antibiotics are used. Here Amp and Cam are among the most used.

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This guide is to use the biolector.
NB! The machine is very slow - be patient!

Materials

  • Biolector

Procedure

  • Turn on the machine (bottom in the back, to the right)
  • Principal window ----> “Monitor”
  • When the lid opens, place the plate in the machine between the metal barrages - it should be firmly secured.
  • For create a new program go to second window “Start …” and follow the steps:
  • Choose new protocol and give it a name (if it is a warm-up program, specify it - the machine needs to warm up for 20-30 min before you add the plate) ----> Next. Names should be written as DDMMYYiGEM_*temperature*_XXXXX
  • Select the type of plate (MTP 48 wells FlowerPlate) ----> Next
  • Choose Layout (48 MTP) ----> Next
  • Select rpm (1000 rpm), temperature and humidity control, and manual (by defect) ----> Next
  • Select the filters to use (Biomass –> Gain 10, PFP –> Gain ?, GFP –> Gain ? ) ----> Next
  • Choose cycle time (30 min for the warm-up program and 3 min (more if several filters are addded) for our program) ----> Start
  • The machine will ask for refilling the deposit with water. Once done, press OK and the program will start running. Be sure to fill up water once a day.
  • When the running is finished, press Stop and save the file in the computer (in the machine, it is saved automatically). You need to press the 'update' button on the computer to get the updated data.
  • In the software go to “Data Management” and select “Transfer the data…”. You can create a new folder or select one already created and press create file. The file will be saved in the folder in cvs format.

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Protocol for preparation and execution of the 1 L bioreactor (Sartorius Stedim 1L) cultivations. .

Materials

Materials

  • 1L Sartorius Stedim 1L bioreactor
  • Sartorius Stedim BIOSTAT Q Plus control tower
  • Off-gas
  • Minimal media
  • Sigma 204 Antifoam
  • Sterile arginine solution
  • Acid (2M Sulfuric acid)
  • Base (2M Potassium hydroxide)
  • Spore suspensions

Consumables

  • 1 mL syringes
  • Lab grease
  • White strips

Procedure

Assembly of the bioreactors

  • Mount the inoculation, sampling, and temperature port from the bottom of the lid and secure them with the bolt from the top of the lid.
  • Mount the impellers at the correct height.
  • Attach the sparger and baffles, and secure them with the bolt at the top of the bioreactor.
  • Mount the condenser, preferably as close to the control tower as possible.
  • The mouth of the condenser is loosely packed with a bit of glass wool, and the condenser is closed off.
  • The temperature and sampling pipe is mounted, and the sampling tubing is attached and sealed off with both a butterfly clamp and a regular clamp.
  • The inoculation port (with membrane) is assembled and mounted.
  • The 4-way connector is attached to the bioreactor, and the un-needed connections are closed off with a knotted piece of tubing.
  • Marprene tube is attached to the air inlet, and an air filter is attached to the tubing in the right direction.
  • Assemble the bioreactor, using a bit of lab grease on the rubber gaskets.
  • Connect the empty 250 mL Blue Cap bottles (acid/base) to the 4-way connector, with tubing appropriate for the control tower pumps.
  • Clamp tubing between the bioreactor and bottles, as close to the reactor as possible. Also, attach a clamp the tube leading to the air filter.
  • Secure all tubing with white strips.
  • Fill the reactor with 1L of minimal medium (see media recipe).
  • Calibrate the pH-electrode, and pressure test the reactor.
  • Cover all open ends and filters with tin foil to protect them during autoclaving.
  • Check all clamps prior to autoclavation, and loosen the main screw to prevent overpressure.
  • Autoclave the bioreactor.
  • After autoclavation, close the loose fitting and mount the air supply. Set the flow fate on the DCU to 0.75 vvm, and remove all clamps except for on the sampling tubing.
  • Once the medium has cooled down, connect the condenser tubing, the temperature jacket tubing, pH-electrode, DO-electrode, and the stirrer.
  • Set the stirrer to 800 r.p.m. and check the accuracy of the pH-electrode. Calibrate if needed.
  • Mount the tubing for the acid and base on their respective pumps, run the acid/base through the tubing and switch to “auto”.
  • Calculate the inoculum volume to a final concentration of 106 spores/mL), and inoculate the bioreactors with the previously prepared Spore suspensions (See Here).
  • Add 50 µL of Sigma 204 Antifoam to the reactor.
  • Due to species limitations, 0.7 g of arginine was added to the media of the positive control.
  • Set the timer to zero at the DCU, and start the batch fermentation at the desired process values.
  • Please find the ramps and process conditions for our bioreactor cultivations below.

Bioreactor conditions

    Process conditions
    Airflow inlet
    Time (min) ValueVVM (L/L/M)
    0 0.1
    300 0.16
    420 0.4
    600 1
    pH
    Time (min) ValuepH
    0 3
    600 5
    Stirrer speed
    Time (min) ValueRPM
    0 100
    300 200
    420 300
    600 500
    720 800
    Temperature
    Time (min) Value(° c)
    Start-end 30

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By David Faurdal, adapted in part from NEB's one-taq protocol.

The purpose of this protocol is to confirm correct insertion of fragments after assemblies, such as 3A, Gibson, or Golden Gate. As the fragments run on the gel won't be used for cloning purposes, there is no reason to use high-fidelity polymerases on this, just use one-taq.

Materials

  • Transformants from whatever assembly method you fashion
  • Eppendorf tubes
  • Sterile toothpicks/inoculation lops/pipette tips for transferring colonies
  • Sterile water
  • LB media with apropriate antibiotics

Procedure

Preparing the template DNA from the transformants:

  • Pick a number of transformants, typically 3-10, from each plate of interest and mark them on the back of the plate.
  • Set up 2 eppendorf tubes for each colony and mark them accordingly:
  • Fill the first one (1) with 15 µL MQ water.
  • The other one (2) remains empty for now.
  • Transfer each colony to the eppendorf containing 15 µL water using a sterile toothpick, inoculation loop or autoclaved pipette tip.
  • Transfer 5 µL of the water from (1) to the empty (2) tube. This tube (2) is now for safekeeping in case the colony PCR shows that the transformant in question contains the correct insertion.
  • Boil the (1) tubes for 10 minutes at 98 °C. Prepare the PCR mastermix, while the colonies are boiling.

Setting up the PCR itself:

  • Set up a 25 µL reaction for each colony to be screened, as per NEB'S one-taq PCR protocol (see the protocolfor troubleshooting).
Component 25 µL reaction
5x OneTaq Standard Reaction Buffer 5 µL
10 mM dNTP 0,5 µL
10 µM Forward Primer 0,5 µL
10 µM Reverse Primer 0,5 µL
OneTaq DNA Polymerase 0.125 µL
Template 1 µL from the (1) tube
Nuclease-Free Water up to 25 µL
  • Run the PCR in the thermocycler (use this website to calculate temperatures used based upon the primers and polymerase used).
  • Run the products on a gel to check for correct insertion.
  • Prepare an O/N culture from the (2) tubes that have the correct insertions by adding 1 mL LB media to the tube, mixing it and transfer a W-tube containing 4 mL LB media.

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The procedure determine the glucose concentration of a sample using 3,5-dinitroalicylic acid (DNS). Adapted by Philip Sørensen and Jacob Mejlsted from D. Navarro et al. and Worthington biochem.

Materials

Materials

  • Plate reader

DNS reagent

  • 1 grams of 3,5-dinitrosalicylic acid
  • Milli-Q water
  • 30.0 grams sodium potassium tartrate tetrahydrate
  • 20 mL 2 M NaOH

Consumables

  • Microtiter plate

Procedure

Preparation of DNS reagent

  • Dissolve 1 g of 3,5-dinitrosalicylic acid in 50 mL Milli-Q water
  • Add slowly 30.0 grams of sodium potassium tartrate tetrahydrate
  • Add 20 mL of 2 M NaOH
  • Dilute to a final volume of 100 mL with reagent grade water. Protect from carbon dioxide and store no longer than 2 weeks.

Running the assay

  • 100 µL media/sample is added to the microtiter plate
  • 100 µL DNS regeant is added to each sample
  • Plate is heated to 98 C for 10 min
  • Absorbance at 540 nm is measured
  • Remember to include a standard to determine actual concentrations

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Adapted by Jacob Mejlsted from NEB's product information and Barrick Lab
This protocol describes digestion of DNA with DpnI. This can be used after a PCR reaction to remove the template DNA. It is recommended that the PCR product is purified before this digestion is conducted, but not necessary.

Materials

Chemicals

  • DpnI enzyme

Procedure

DpnI digestion

  • Add 1 µL of DpnI to finished 50 µL PCR reactions (or .5 µL to 25 µL reactions). Pipet or invert to mix.
  • Incubate the mixture at 37 °C for 1-2 hrs.
    Alternatively, the solution can be left overnight at room temperature. Periodic mixing may aid digestion (but is unnecessary).
  • PCR cleanup or gel-purify the reaction for downstream processes OR heat inactivate at 80 °C for 20 min.

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Adapted by Jacob Mejlsted & Kyle Rothschild

Materials

Equipment

  • Autoclave
  • Freezer
  • Centrifuge
  • Spektrophotometer

Consumables

  • PCR tubes
  • 50 mL falcon tubes

Chemicals

  • MgCl2
  • CaCl2
  • SOC/SOB
  • Glycerol

Procedure

Day 1:

  • Autoclave the following:
    • Minimum 200 mL 0.1 M MgCl2
    • Minimum 150 mL 0.1 M CaCl2
    • Minimum 100 mL 85 mM CaCl2 + 15% glycerol (v/v)
    • Minimum 700 mL SOC
    • 3 x shakeflasks (500 mL)
  • Freeze at -20 degC (after autoclaving)
    • 0.1 M MgCl2
    • 0.1 M CaCl2
    • 85 mM CaCl2 + 15% glycerol (v/v)
    • Minimum 10 x 50 mL falcon tubes
    • PCR tubes

Day 2:

  • Culture growth
  • Early in the morning, start cooling the Sorval centrifuge to 4 C
  • Pour 200 mL SOB media into each of the shakeflask (one shakeflask per starter culture).
  • Mark the shakeflasks to match the startercultures
  • Measure the OD 600 of each starterculture, and inoculate the shakeflask with a volume so the final OD 600 value in the shakeflask culture becomes 0.01
  • Grow the shakeflask culture at 37 C with shaking. Measure OD values of the sample every 20 minutes once the OD 600 value is above 0.2.
  • When OD 600 is between 0.3 and 0.4, put the cultures into an ice bath immediately, and swirl the shakeflash around in the cold water to cool culture. Chill the culture in the icewater for 20-30 minutes, occasionally swirling the cultures.
  • FROM THIS STAGE ON, KEEP CELLS AT ICE/4 °C AT ALL TIMES
  • For each shakeflask culture, pour the culture into 3 x 50 mL frosted falcon tubes from the freezer.
  • Keep the tubes on ice
  • Centrifuge falcon tubes at 3000 x g for 15 min at 4 C (Spin #1 of 4)
  • Discard supernatant, and resuspend cells in 15 mL icecold 0.1 M MgCl2
  • Keep tubes with cells on ice
  • Pool the resuspended cells into one of their matching falcon tubes, so you now have 3 different 50 mL falcon tubes, one with cells corresponding to each of the starter cultures you had.
  • Keep tubes on ice
  • Centrifuge falcon tubes at 2000 x g for 15 min at 4 o C (Spin #2 of 4)
  • Discard the supernatant, and resuspend pellet in 40 mL icecold 0.1 M CaCl2
  • Keep tubes on ice
  • Let cell suspensions stand in ice for 20-30 minutes
  • Centrifuge falcon tubes at 2000 x g for 15 min at 4 C (Spin #3 of 4)
  • Discard supernatant, and resuspend pellet in 10 mL icecold 85 mM CaCl 2 + 15 % glycerol
  • Keep tubes on ice
  • Centrifuge falcon tubes at 1000 x g for 15 minutes at 4 C (Spin #4 of 4)
  • Pellet might look small and will be a bit fragile. Handle tubes with care when taking them out of centrifuge
  • Attention: The next few steps are best done on ice inside a LAF bench
  • Resuspend pellet in 800 µL ice-cold 85 mM CaCl2 + 15 % glycerol
  • Put falcon tubes on ice
  • Immediately after cells are confirmed resuspended, aliquot 30 µL of the competent cell culture into the chilled PCR tubes
  • Put tubes into -80 °C freezer as fast as possible

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Here, a description of the instruments, settings, and general protocol for the fluorescence measurements can be found. .

Materials

Materials

  • SpectraMax iD3

Consumables

  • Corning® Clear Polystyrene 96-Well Microplate

Procedure

Fluorescence measurements

  • If not done already, 100 µL of sample was loaded into the well of a spectophotometry plate.
  • The RFP was measured using the following settings: (Ex/Em)
    • RFP: 584/607
    • GFP: 485/510

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Protocol for PCR & Gibson adapted from the Gibson Assembly Cloning Guide 2nd edition
Protocol for template removal adapted from NEBcloner
Protocol for PCR Purification adapted from Qiagen QIAquick® PCR Purification Kit
Protocol for MiniPrep adapted from Qiagen's QIAprep® Spin Miniprep Kit

Procedure

PCR Amplfication of DNA Fragments

  • Prepare PCR reaction (see table)
Component Volume
Insert or vector DNA (100 pg/µL - 1 ng/µL in TE) 0.5 µL
10 µM Forward Primer 2.5 µL
10 µM Reverse Primer 2.5 µL
10 mM dNTPs 1 µL
5X Phusion HF Buffer 10 µL
Phusion DNA Polymerase (2 U/µL) 0.5 µL
Nuclease-free Water 33 µL
Total 50 µL
  • Run reaction in a thermocycler
Step Temperature Duration Number of Cycles
Initial denaturation 98 °C 30 seconds 1 cycle
Amplification 98 °C 10 seconds 25-30 cycles
Primer Tm 20 seconds
72 °C 30 seconds/kb
Final extension 72 °C 5 minutes 1 cycle
Hold 4 °C - 1 cycle

Template Removal

  • Set up the reaction as follows:
Component 50 µL Reaction
Component 50 µL Reaction
DNA 1 µg
10X CutSmart Buffer 5 µL (1X)
DpnI 1.0 µL (or 10 units)
Nuclease-free Water to 50 µL
  • Incubate at 37 °C for 5–15 minutes (DpnI is Time-Saver qualified.)
  • Optional: Heat inactivate at 80 °C for 20 min if not doing a DNA purification step

PCR Purification

  • Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10 µL 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow
  • Place a QIAquick column in a provided 2 mL collection tube
  • To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s. Discard flow-through and place the QIAquick column back in the same tube.
  • To wash, add 750 µL Buffer PE to the QIAquick column centrifuge for 30–60 s. Discard flow-through and place the QIAquick column back into the same tube.
  • Centrifuge the QIAquick column once more in the provided 2 mL collection tube for 1 min to remove residual wash buffer.
  • Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.
  • To elute DNA, add 50 µL Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µL elution buffer to the center of the QIAquick membrane, let the column stand for 1 min and then centrifuge.

Mid-way analysis

  • Add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
  • Run the gel and evaluate the results
  • NanoDrop the purified DNA to determine the concentration

Gibson Assembly using a HiFi 1-Step Kit

  • Thaw Gibson Assembly HiFi 1-step Master Mix on ice
  • Combine insert and vector DNA to a total volume of:
    5 µL for the 2X kit or
    7.5 µL for the HC 4X kit
  • Vortex the master mix
  • On ice, combine
    5 µL DNA and 5 µL Master Mix for the 2X kit or
    7.5 µL DNA and 2.5 µL Master Mix for the HC 4X kit
    Mix well andbriefly centrifuge
  • Incubate at 50 °C for 1 hour
  • Store reactions at -20 °C or use in 1-2 µL per reaction for transformation

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This protocol is made to assembly parts with TypeIIs enzyme BsaI HF v2. These parts will most probably have the standard Moclo overhangs. Made by PHS, adapted from here.

Materials

Reagens

  • T4 DNA ligase
  • 10X T4 DNA ligase buffer
  • BsaI HF v2 enzyme (NEB)
  • Reciever plasmid (BsaI compatible like pGIA2P2o )
  • DNA fragments with BsaI overhangs
  • Milli-Q water

Media

Materials

  • PCR tubes
  • 10 µL pipette tips
  • Thermocycler

Procedure

Setting up the reaction

  • In a PCR tube, mix the following:
    - 0.5 µL of T4 DNA Ligase
    - 2 µL of 10X T4 DNA Ligase Buffer
    - 0.5 µL of BsaI HF v2 restriction enzyme
    - 100 ng of receiver plasmid
    - Equimolar amounts of inserts
    - Milli-Q for a total volume of 20 µL.
  • Mix gently
  • Place the tube on a thermocycler

Thermocycler program

Step Temp Time
Activation of BsaI HF v2 37 °C 5 min
Activation of T4 ligase 16 °C 5 min
Repeat step 1 & 2 for 25 cycles
Inactivation BsaI HF v2 65 °C 20 min
Inactivation T4 ligase 85 °C 10 min
Hold 4 °C Hold

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This protocol is made to assembly parts with TypeIIs enzyme SapI

Materials

Reagens

  • T4 DNA ligase
  • 10X T4 DNA ligase buffer
  • SapI enzyme (NEB)
  • Reciever plasmid (Sap one compatible like ​Inaccessible DNA Sequence )
  • DNA fragments with SapI overhangs
  • Milli-Q water

Reagens

  • PCR tubes
  • 10 µL pipette tips
  • Thermocycler

Procedure

Setting up the reaction

  • In a PCR tube, mix the following:
    - 0.5 µL of T4 DNA Ligase
    - 2 µL of 10X T4 DNA Ligase Buffer
    - 0.5 µL of Type IIS restriction enzyme
    - 100 ng of receiver plasmid
    - Equimolar amounts of inserts
    - Milli-Q for a total volume of 20 µL.
  • Mix gently
  • Place the tube on a thermocycler

Thermocycler program

Step Temp Time
Activation of SapI 37  °C 5 min
Activation of T4 ligase 16  °C 5 min
Repeat step 1 & 2 for 25 cycles
Inactivation SapI 65  °C 20 min
Inactivation T4 ligase 85  °C 10 min
Hold 4  °C Hold

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Adapted by Jacob Mejlsted & Joen Haahr Jensen from IDT's HiFi assembly protocol.

Materials

(X is the number of reactions)

Consumables

  • X PCR tubes for each reaction + 1 for positive control
  • X eppendorf tupe for each reaction + 1 for postive control
  • X selection plate for each reaction
  • 1 Amp plate for positive control

Chemical

  • Hifi DNA assembly Master mix
  • Sterile Milli-Q water
  • Competent E. coli cells
  • Prepared DNA fragments for assembly

Procedure

Assembly protocol

  • Set up the following reaction on ice:
    * If the inserts are less less than 200 bp, use a 5 fold excess of inserts instead of a 2 fold excess
    ** If a greater number of fragments are assembled, increase the volume of the reaction and use additional HiFi DNA assembly master mix
Recommended amount of fragments used for assembly
2-3 Fragments* 4-6 Fragements** Postive control
Recommended DNA Molar Ratio Vector:insert = 1:2 Vector:insert = 1:1
Total amount of DNA fragments 0.03-0.3pmols
X µL		 0.2-0.5 pmols
X µL		 10 µL
NEB Hifi Assembly master mix 10 µL 10 µL 10 µL
Milli-Q water 10-X µL 10-x µL 0 µL
Total volume 20 µL 20 µL 20 µL
  • Incubate the reaction samples in a thermocycler at 50 °C for 15 minutes (when 2-3 fragments are assembled) or 60 minutes (when 4-6 fragments are assembled). Following incubation, store the reaction samples at -20 °C for subsequent transformation
    Note: Extended incubation up to 60 minutes can in some cases improve transformation efficiency

Transformation protocol

  • Thaw chemically-competent cells on ice
  • Add 2 µL of the chilled assembly product to the competent cells. Mix gently by pipetting up or down or by flicking the tube 4-5 times. Do NOT vortex
  • Place the mixture on ice for 30 minutes. Do not mix
  • Heat shock at 42 °C for 30 seconds. Do not mix
  • Transfer tubes to ice for 2 minutes
  • Add 950 µL of room temperature SOC media to the tubes
  • Incubate the tube for 37 °C for 60 minutes. shake vigerously (250 rpm) or rotate
  • Warm selection plates to 37 °C
  • Spread 100uL of the cells onto the selection plates.
  • Note: Use Amp plates for the positive control
  • Incubate overnight at 37 °C.

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Protocol for production of 1 liter of minimal media

Materials

Materials

  • 50 mL of 20% w/V D-glucose stock
  • 50 mL of 20x nitrate salts stock
  • 1 mL 1000x Trace elements stock
  • 1 mL of 1% thiamine stock
  • 20 g agar

Procedure

Media preparation

  • Mix ingridients in flask and add MiliQ water to 1 litre
  • Mix with magnet stirrer until the sugar is dissolved
  • Autoclave at 121 °C for 20 min

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Here is a list of miscellaneous protocols, all of which were performed according to the manufacturer's and/or the referenced specifications. .

Protocols

Gel electrophoresis

  • Gels contained 1% agarose and were stained with 1000X SYBR Safe (Invitrogen)
  • DNA fragment solutions were combined with 6X Purple Loading Dye (New England Biolabs).
  • Samples were run in 1X TAE running buffer at 100V with a 1kb DNA ladder (New England Biolabs).
  • Electrophoresis was run on the RunOne Electrophoresis System (Embi Tec).
  • Pictures of the resulting gels were taken with the ChemiDoc™ XRS+ Imaging System or equivalent.

Gel purifications

  • Isolated DNA bands were purified via the QIAquick Gel Extraction kit (QIAGEN).

PCR spin purifications

  • When deemed necessary, plasmid DNA was spin purified, rather than gel purified, via the QIAquick PCR Purification Kit (QIAGEN).

Plasmid miniprep purification

  • Plasmids were purified using the QIAprep Spin Miniprep Kit (QIAGEN).

DNA sequencing

  • DNA was verified by Sanger sequencing, using the Eurofins’ overnight sanger sequencing service.

Primer/DNA resuspension

  • Primers were diluted to a stock concentration of 100 µM and a working concentration of 10 µM.
  • Synthetic DNA (e.g. synthetic promoters) were diluted to 25 ng/µL or 40 ng/µL, depending on downstream uses.

This protocol is for linearising backbones with the PacI+Nt.BbvCI USER casette. This will allow assembly of PCR product with USER tails to be assembled into this linearised plasmid

Materials

For digest I

  • Plasmid: 40 µL
  • PacI: 2 µL
  • Cutsmart buffer (NEB): 10 µL
  • MQ water: 48 µL

For digest II

  • Finished digest I
  • Nt.BbvCI: 2uL

Procedure

Digest I

  • MIx all the reagents as mentioned above, and incubate at 37 °C overnight

Digest II

  • Take the digest I reaction from the 37 °C incubator, add in the 2 µL Nt.BbvCI to the reaction mixture, and incubate for 2 hours at 37 °C
  • Heat inactivate the samples at 80 °C for 30 min

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Protocol for PCR using the Phusion polymerase adapted by Jacob Mejlsted from the NEB protocol for the same product.

Materials

DNA

Consumables

  • PCR tubes (1 per reaction + 1 for positive control)

Chemicals

  • Forward primers
  • Reverse primers
  • Nuclease-free water
  • Phusion DNA Polymerase
  • 5X Phusion HF or GC Buffer
  • 10 mM dNTPs
  • DMSO (optional)

Procedure

PCR Amplfication of DNA Fragments

  • Prepare PCR reaction (see table)
Component 20 µL Reaction 50 µL Reaction Final Concentration
Nuclease-free water to 20 µL to 50 µL
5X Phusion HF or GC Buffer 4 µL 10 µL 1X
10 mM dNTPs 0.4 µL 1 µL 200 µM
10 µM Forward Primer 1 µL 2.5 µL 0.5 µM
10 µM Reverse Primer 1 µL 2.5 µL 0.5 µM
Template DNA variable variable < 250 ng
DMSO (optional) (0.6 µL) (1.5 µL) 3%
Phusion DNA Polymerase 0.2 µL 0.5 µL 1.0 units/50 µL PCR
  • Alternatively, a master mix can be prepared
Reactant Per reaction (50uL) [µL] Mastermix [µL]
Number of reactions 1 10
5X Phusion HF or GC Buffer 10 100
10 mM dNTPs 1 10
10 µM Forward Primer 2.5 Added individually
10 µM Reverse Primer 2.5 Added individually
Template DNA variable Added individually
Phusion DNA Polymerase 0.5 5
DMSO (optional) 0 0
Milli-Q 33.5 335
  • Run reaction in a thermocycler
    *15 seconds/kb works for most reactions. 30 seconds/kb can be used for more complex reaction, such as cDNA.
Step Temperature Duration Number of Cycles
Initial denaturation 98 C 30 seconds 1 cycle
Amplification 98 C 10 seconds 25-30 cycles
Primer Tm 20 seconds
72 C 15-30 seconds/kb*
Final extension 72 C 5-10 minutes 1 cycle
Hold 4 C - 1 cycle

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Adapted by Jacob Mejlsted from the NEB protocol for the same product.

Materials

  • 1 PCR tube per reaction

Chemicals

  • OneTaq standard reaction buffer
  • dNTPs
  • Primers
  • DNA template
  • OneTaq DNA Polymease

Procedure

Method

  • To a tube add the following
Component 25 μL reaction*** 50 μL reaction*** Final Concentration
5X OneTaq Standard Reaction Buffer* 5 µL 10 μL 1X
10 mM dNTPs (#N0447) 0.5 µL 1 μL 200 µM
10 µM Forward Primer 0.5 µL 1 μL 0.2 µM
10 µM Reverse Primer 0.5 µL 1 μL 0.2 µM
OneTaq DNA Polymerase 0.125 µL 0.25 µL 1.25 units/50 µL PCR**
Template DNA** variable variable < 1,000 ng
Nuclease-free water to 25 µL to 50 µL

*OneTaq GC Reaction Buffer and High GC Enhancer can be used for difficult amplicons
**For plasmids or viral DNA, use 1 pg–10 ng DNA for a 50 µL reaction.
***It can be advantagous to pool some of the parts into a master mix, as some labs cannot dispense 0.125 µL accurately.

  • Alternatively, a master mix can be prepared
Reactant Per reaction (50uL) [µL] Mastermix [µL]
Number of reactions 1 10
5X OneTaq Standard Reaction Buffer* 10 100
10 mM dNTPs (#N0447) 1 10
10 µM Forward Primer 1 Added individually
10 µM Reverse Primer 1 Added individually
OneTaq DNA Polymerase 0.25 2.5
Template DNA 1 Added individually
  • Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary.
STEP TEMP TIME
Initial Denaturation 94 °C 30 seconds
30 Cycles 94 °C 15-30 seconds
45-68 °C 15-60 seconds
68 °C 1 minute per kb
Final Extension 68 °C 5 minutes
Hold 4-10 °C
  • PCR products can then by digested by DpnI, purified, stored at -20 C, or a combination.

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Protocol for PCR using the Q5 2X master mix adapted by Jacob Mejlsted from the NEB protocol for the same product.

Materials

DNA

Consumables

  • PCR tubes (1 per reaction + 1 for positive control)

Chemicals

  • Forward primers
  • Reverse primers
  • Nuclease-free water
  • Q5 2X Master Mix

Procedure

PCR Amplfication of DNA Fragments

  • Prepare PCR reaction (see table)
Component 25 µL reaction 50 µL reaction
Q5 High-Fidelity 2X Master Mix 12.5 µL 25 µL
10 µM Forward Primer 1.25 µL 2.5 µL
10 µM Reverse Primer 1.25 µL 2.5 µL
Template DNA* variable (0ften 0.5 µL) variable (0ften 1 µL)
Nuclease-Free Water to 25 µL to 50 µL
Total 25 µL 50 µL

*Template DNA:
For a 50 µL reaction, 1 ng-1 µg is recommended for genomic DNA and 1 pg-10 ng is recommended for plasmid or viral DNA.

  • Run reaction in a thermocycler
Step Temperature Duration Number of Cycles
Initial denaturation 98 C 30 seconds 1 cycle
Amplification 98 C 10 seconds 25-30 cycles
Primer Tm 20 seconds
72 C 30 seconds/kb
Final extension 72 C 2 minutes 1 cycle
Hold 4 C - 1 cycle
  • The PCR products can then be stored at -20 C, used directly, or purified using PCR purification or gel extraction

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This protocol describes the required components and method for a X7 polymerase mediated PCR reaction. .

Materials

Reagents

    COMPONENT 50 µL REACTION
    CXL buffer 10 µL
    10 mM dNTPs 1 µL
    10 µM Forward Primer 2.5 µL
    10 µM Reverse Primer 2.5 µL
    Template DNA Variable (often 1 µL)
    Pfu X7 Polymerase 0.5 µL
    DMSO 1.5 µL
    Nuclease-Free Water to 50 µL

Consumables

  • PCR strip/tubes

Procedure

Thermocycler PCR program

    Step Temperature Duration Number of Cycles
    Initial denaturation 95 C 2 minutes 1 cycle
    Amplification 95 C 30 seconds 25-30 cycles
    Primer Tm 30 seconds
    72 C around 1 min/kb*
    Final extension 72 C 5 minutes 1 cycle
    Hold 4 C - 1 cycle

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This protocol is an adaptation of a GUS (β-glucuranidase) assay provided by Zofia Dorota Jarczynska and adapted by Jacob Mejlsted

Materials

Extraction buffer (1L)

  • 50 mM Na3PO4, pH 7 (50 mL of 1 M stock solution)
  • 10 mM β-mercaptoethanol (0.7 mL of 14.4 M stock solution)
  • 10 mM Na2EDTA (20 mL of 0.5 M Na2EDTA stock solution)
  • 0.1% Sodium Lauryl Saccosine (10 mL of 10% Sarcosyl)
  • 0.1% Triton X-100 (10 mL f 10% Triton)
  • Milli-Q water up to 1L

Equipment

  • Centrifuge with cooling
  • Plate reader
  • Homogenizer
  • Beads (big, metal)
  • Sterile funnels with mira cloth

Chemicals

  • Bradford reagent (Like SigmaAldrich B6916)
  • BSA standard (Bovine Serum Albumin 2 mg/mL)

Procedure

Protein Extraction

  • CRITICAL Set the centrifuge at 4 °C
  • Seperate biomass from supernantant using mira cloth or other methods
  • Take ~100mg biomass and place in a FastPrep tube with one big metal bead
  • Place the tubes in liquid nitrogen
  • Homogenize for 1 min at 45 Hz
  • Add 500 µL extraction buffer
  • Homogenize for 2 min at 45 Hz
  • Centrifuge for 2 min at 10,000 x g at 4 °C
  • Collect the liquid phase and place in a new tube
  • Centrifuge for 15 min at 10,000 x g at 4 °C
  • Aliquote the supernantant into new tubes
  • Store at -80 °C

Bradford Assay

  • Transfer 100 µL protein sample into a microtiter place
  • Measure fluorescense at the appripriate wavelenghts

Fluorescence Assay (optional)

  • Mix 20 µL protein sample with 230 µL Bradfor reagent
  • Transfer to a new microtiter plate
  • Measure the blue color at 595 nm
  • Remember to include a standard curve and Bradford reagent
  • Determine protein concentration

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Adapted by Jacob Mejlsted from iGEM's protocol

Materials

Materials

  • Resuspended DNA to be transformed
  • 10 pg/µL Positive transformation control DNA (e.g. pSB1C3 w/ BBa_J04450, RFP on high-copy chloramphenicol resistant plasmid. Located in the Competent Cell Test Kit.)
  • Competent Cells (50 µL per sample)
  • 1.5 mL Microtubes
  • SOC Media (950 µL per sample)
  • Petri plates w/ LB agar and antibiotic (2 per sample)

Equipment

  • Floating Foam Tube Rack
  • Ice & ice bucket
  • Lab Timer
  • 42 °C water bath
  • 37 °C incubator
  • Sterile spreader or glass beads
  • Microcentrifuge

Procedure

Method: Day 1

  • Resuspend DNA in selected wells in the Distribution Kit with 10 µL dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
  • Label 1.5 mL tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 1.5 mL tubes (one tube for each transformation, including your control) in a floating foam tube rack.
  • Thaw competent cells on ice: This may take 10-15 min for a 260 µL stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
  • Pipette 50 µL of competent cells into 1.5 mL tube: 50 µL in a 1.5 mL tube per transformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Don’t forget a 1.5 mL tube for your control.
  • Pipette 1 µL of resuspended DNA into 1.5 mL tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.
  • Pipette 1 µL of control DNA into 2 mL tube: Pipette 1 µL of 10 pg/µL control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
  • Close 1.5 mL tubes, incubate on ice for 30 min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
  • Heat shock tubes at 42 °C for 45 sec: 1.5 mL tubes should be in a floating foam tube rack. Place in water bath to ensure the bottoms of the tubes are submerged. Timing is critical.
  • Incubate on ice for 5 min: Return transformation tubes to ice bucket.
  • Pipette 950 µL SOC media to each transformation: SOC should be stored at 4 °C, but can be warmed to room temperature before use. Check for contamination.
  • Incubate at 37 °C for 1 hours, shaking at 200-300 rpm
  • Pipette 100 µL of each transformation onto petri plates Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
  • Incubate transformations overnight (14-18hr) at 37 °C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.

Method: Day 2

  • Pick single colonies: Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and miniprep.
  • Count colonies for control transformation: Count colonies on the 100 μL control plate and calculate your competent cell efficiency. Competent cells should have an efficiency of 1.5x108 to 6x108 cfu/µg DNA.

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Here, the procedure for sampling of the shake-flask and bioreactor cultivations is described.

Materials

Consumables

  • 10 mL syringe (bioreactor)
  • 15 mL falcon tube
  • 1.5 mL eppendorf tube
  • Corning® Clear Polystyrene 96-Well Microplate

Procedure

Shake flask cultivation

  • In a clean bench, approximately 1 mL of sample containing biomass was sampled via pipetting.
  • 100 µL sample containing biomass was transferred to a spectrophotometry plate.
  • 200 µL of sample was spun down at 10.000 g for 3 min. and 100 µL transferred to a spectrophotometry plate.
  • Immediately after sampling, samples in the spectrophotometry plate was measured for fluorescence according to the MEASUREMENT PROTOCOL, and the remainder of the sample in the eppendorf tubes stored at -20 C.

Bioreactor cultivation

  • For sampling of the bioreactor, extra care was taken to prevent contamination.
  • The sterile 10 mL syringe was sprayed with 70% ethanol and inserted in the sampling tube.
  • The clamps on the sampling tube were loosened and 5 mL sample was drawn and discarded.
  • The syringe was again sprayed with 70% ethanol, and reinserted in the sampling tube.
  • Approximately 10 mL sample containing biomass was drawn and transferred to a 15 mL falcon tube.
  • The clamps on the sampling tube were tightened, and the sampling tube sprayed with 70% ethanol.
  • The samples were stored at -20 C for later protein purification.

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Here, we describe the protocol for the set-up and execution of the shake flask cultivations. Please, click here) for the media recipe, here for sampling procedures for the specifics regarding sampling, and fluorescence measurements for details regarding the RFP measurements.

Materials

Materials

  • Pyrex shake-flasks (with baffles)
  • Minimal media
  • Spore suspension

Consumables

  • Cotton stopper
  • Tin foil

Procedure

Preparations and inoculation

  • A breathable cotton ball was added to the mouth of a glass shake-flask (with baffles), covered with tin foil, and then autoclaved.
  • 150 mL minimal media was added to each shake-flask.
  • The shake-flasks were inoculated with their respective spore solutions to a final concentration of 106 spores/mL.
  • Shake-flasks were incubated for 5 days as described below.

Conditions

  • Shake-flasks were incubated at 30 C at 150 rpm for 5 days.
  • Samples were taken as regularly as described in sampling procedures.

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The following is a quick and easy protocol for creating the spore suspensions used for storage and the inoculation of the shake-flask, biolector, and bioreactor cultivations. .

Materials

Consumables

  • 15mL falcon tube
  • 5mL syringe

Chemicals

  • H2O + 0.01% tween

Procedure

Creating the spore suspensions

  • An Aspergillus strain is grown up until fully sporulated.
  • ~5 mL of H2O + 0.01% is deposited via the 5 mL syringe onto a fully sporulated plate of Aspergillus
  • With the needle, spores are dissolved in the liquid and the spore suspension placed into the 15 mL falcon tube.
  • The spore suspension can be kept for up to 3 months at 4 C.

Calculating spore concentration and adjusting

  • Before use, the spore concentration is determined via. microscopi.
  • By further dilution with H2O + 0.01%, the spore suspension is adjusted to approximately 109.

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Protocol for production of 1 liter of fungal transformation media

Materials

  • 342,30 g Sucrose
  • 50.0 mL Nitrate salts
  • 1 mL Trace metal elements
  • 1 mL Thiamine
  • 21 g Bacto agar

Procedure

  • Mix ingridients in flask and add MiliQ water to 1 litre.
  • Mix with magnet stirrer until the sugar is dissolved.
  • Autoclave at 121 °C for 20 min.

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The following describes the USER-cloning protocol used during our project.

Materials

Reagents

  • USER-linearized insert(s)
  • USER-linearized backbone
  • USER enzyme
  • CutSmart buffer
  • Milli-Q water

Consumables

  • PCR-strip/tubes

Procedure

Preparing USER-reaction

  • In a PCR-tube, combine;
    • 200 ng USER-linearized insert(s)
    • 100 ng USER-linearized vector backbone
    • 1 µL 10X CutSmart buffer
    • 1 µL USER enzyme
    • H2O to 10 µL.
  • Incubate reaction mix as described below.

Incubating reaction mix

  • In a PCR-machine, incubate as follows.
    • 37C for 25 min
    • 25C for 10 min
    • 20C for 10 min
    • 15C for 10 min
    • 10C until further use
  • Transform reaction mixture in E. coli as described in the Hifi assembly protocol

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The logos of our three biggest supporters, DTU Blue Dot, Novo Nordisk fonden and Otto Mønsted fonden The logos of all of our sponsors, DTU, BioNordica, Eurofins Genomics, Qiagen, NEB New England biolabs, IDT Integrated DNA technologies and Twist bioscience