Experiments

Introduction

This page presents the collection of wet lab protocols that our team used through the busy working summer. The purpose of this page is to present all the steps and reagents needed in the protocols in detail. To see our weekly wet lab progress, head over to the Notebook page. To see the final results in detail, please head over to the Results page. To learn more about our engineering approach, head over to the Engineering page.

In this protocol, you will find the step-by-step guide on how to create competent E. coli DH5α cells. Make sure to keep the buffers in the fridge (4°C) before starting the experiment!

Reagents

TYM medium

20 g/L bacto-tryptone
5 g/L Yeast extract
0.1 M NaCl
10 mM MgSO₄

TfB I

30 mM KAc
50 mM MnCl₂ (sterilize through filter, add later)
0.1 M KCl
10 mM CaCl₂
15% glycerol

TfB II

10 mM Na-MOPS, pH=7
75 mM CaCl₂
10 mM KCl
15% glycerol

Protocol

Start ON culture in TYM medium (5mL).
On the next morning, measure OD₆₀₀. Calculate how much to take from the ON culture with the formula: OD₆₀₀ X V = 0.08 X 300mL.
Inoculate 300 mL of TYM medium with the ON culture at OD₆₀₀=0.08 (200 rpm).
Grow cells to OD₆₀₀=0.6 (around 1.5 hours). Take measurements each half hour until the culture reaches the desired OD₆₀₀.
Cool cell cultures on ice (keep cells cold from now on).
Transfer culture in 50 mL tubes.
Centrifuge 15 min., GSA rotor, 4200 rpm, 4°C.
Discard supernatant, repeat steps 6 and 7 until the whole culture is spun down.
Resuspend pellet in 60 mL of cold TfB I (on ice).
Centrifuge 10 min., GSA rotor, 4200 rpm, 4°C.
Resuspend pellet in 12 mL of cold of TfB II (on ice).
Aliquot 300 μl in Eppendorf tubes.
Freeze in liquid nitrogen (optional).
Store at -80.

In this protocol, you can find how to transform E. coli cells (how to insert the desired plasmid into E. coli) from competent E. coli cells aliquots. Approximate time of the experiment: 2h + overnight incubation.

Materials

Ice
Prewarm heat block at 42ºC
Selective plates with the corresponding antibiotics
Competent cells
Plasmid DNA

Protocol

Defrost 150 µl aliquot of E. coli competent cells per transformation on ice for 15 min. Label one 1.5 mL microcentrifuge tube for each transformation and then pre-chill by placing the tubes on ice.
Add 1 μl plasmid DNA (10 pg/μl), incubate 15 min on ice.
Heat shock the cells for 1 min at 42°C.
Immediately place the cells on ice for 3 min.
Add 1mL LB and incubate at 37°C for 1 hour (200rpm).
Plate 100 μl on selective plates.
Incubate plates ON at 37ºC.

Protocol for Lithium Acetate Yeast Transformation.

Reagents

PLATE Mixture

90mL Sterile 45% PEG 4000
10mL 1M LiOAc
1mL 1M Tris-Cl (pH 7.5)
0.2mL 0.5M EDTA

Other materials

A solution of 10mg/mL of carrier DNA
YPD (Yeast Extract Peptone Dextrose) media
Selective plates
1-2µg of Plasmid in ~5µL of water
Heat block prewarmed at 42ºC

Protocol

Inoculate 2 mL cultures for each transformation in YPD and grow ON at 30ºC.
In an Eppendorf tube, spin down 1mL of cells (5 seconds, 5000 rpm) for each transformation.
Decant the supernatant leaving behind around ~50 μl of the liquid more or less. Resuspend gently.
Add 2 μl of 10mg/mL carrier DNA, resuspending cells with pipet tip.
Add 5 μl of plasmid and vortex.
Add 500 μl of PLATE mixture and vortex.
Add 20 μl of 1.0M DTT and vortex.
Incubate on your benchtop for at least 3 hours (could go up to 24h). Cells will have settled to the bottom of the tube.
Heat shock the cells for 10 min at 42ºC.
Place the pipet tip directly into the bottom of the tube and withdraw 100 μl of cells in PLATE mixture (you will get ~75% of the cells in this way).
Plate cells on selective media and incubate at 30ºC for 2 days.

Notes

Cells can be transformed directly from a plate by taking a toothpick of cells and resuspending them in PLATE mix. The transformation from a fresh plate is very efficient, but will still work from an old (several months) plate.
Factors that improve efficiency: 1. Late log-phase cells yield more transformants than stationary phase cells 2. Heat shock is not necessary but improves efficiency by 5-fold 3. DTT is not necessary but greatly improves efficiency. Use more DTT (up to 100 µL) to further increase efficiency Approximate time for the experiment: Overnight culture + 4-5h (3h min for incubating) + overnight incubation (around 2 days).

Here you can find how to introduce a part into a level 0 vector (pYTK001). Part plasmids are assembled via a BsmBI Golden Gate reaction into the part entry vector. The entry vector contains a ColE1 origin of replication, a chloramphenicol resistance cassette and a GFP expression dropout for green/white screening. The number of DNA inserts can optionally be normalized to equimolar concentrations (∼20 fmol each) to improve assembly efficiencies.

Reagents

4 μL of DNA insert (10 ng/μl)
1 μL of Plasmid (20 ng/μl)
1 μL T4 DNA Ligase buffer
0.5 μL T4 Ligase
0.5 μL BsmBI (10000 U/mL)
Water up to the final volume of 10 μL

Protocol

Step	Temperature (C°)	Time
Digestion	42	2 min
Ligation	16	5 min
Final digestion	60	10 min
Heat inactivation	80	10 min
Hold	4	∞

In this protocol, you can find how to “build” the final cassette plasmid from the part plasmids. After this digestion and ligation, the transformation into E. coli is required to acquire high yields of the plasmid. The cassette plasmid contains a kanamycin resistance cassette.

Reagents

0.5 μL of part 1-8 plasmids
0.5 μL of cassette plasmid
1 μL T4 DNA Ligase buffer
0.5 μL T4 Ligase
0.5 μL BsaI (10000 U/mL)
Water up to the final volume of 10 μL

Protocol

Step	Temperature (C°)	Time
Digestion	42	2 min
Ligation	16	5 min
Final digestion	60	10 min
Heat inactivation	80	10 min
Hold	4	∞

In this protocol you can find how to amplify the G-Blocks using a 2xPhirePol Green Hot Start Mix mastermix.

Reagents

Reagent	Volume (μl)
Master mix	25
Fw primer (10µM)	2.5
Rv primer (10µM)	2.5
Template (50ng)	1
MilliQ	Up to 50

Protocol

Step	Temperature (C°)	Time
Initial denaturation	98	1 min
Denaturation	98	10s
Annealing	60	10s
Extension	72	30s
Final extension	72	1 min
Hold	4	∞

In this protocol you can find how to perform gel electrophoresis for analysis of PCR results or digestion of plasmids results.

Reagents

Agarose
TAE buffer (1x)
Gel electrophoresis set-up

Protocol

To make 1% agarose gel:

500 ml TEA buffer
5g agarose
Melt in the microwave until the mixture is clear

To run the gel electrophoresis:

Pour the solution into a gel cast and add 5μl of SYBR green(Invitrogen SYBR Safe DNA gel stain) to the solution. Let the gel set with a comb for slots inserted. Note: SYBR green is light sensitive
Add 3 μl of ladder (Quick-Load Purple 1kb Plus DNA Ladder, NEB) to one of the slots.
Add 1 μl loading dye (Gel Loading Dye Purple (6x), NEB) to the samples.
Run the gel at 120V for 30-40 min.
Analyse the gel using a GelDoc (Biorad).

In this protocol you can find how to prepare the stock (50x) TAE buffer to run agarose gels.

Reagents

Chemical	M_W	Concentration	Amount
Tris Base	121.1 g/L	2 M	242.2 g/L
Acetic Acid	60.5 mL/L	1 M	60.5 mL/L
EDTA Sodium salt dihydrate	372.24 g/L	50 mM	18.612 g/L

Protocol

Prepare a 500 mM EDTA solution using 93.05 grams of EDTA disodium salt (MW=372.24 g/mol). Dissolve in 400 ml deionized water and adjust the pH NaOH. EDTA will not go completely into solution until the pH is adjusted to about 8.0!
A 50X stock solution is prepared by dissolving 242 g Tris base in 500 mL water, adding 57.1 mL glacial acetic acid, and 100 mL of 500 mM EDTA (pH 8.0) solution, and bringing the final volume up to 1 liter.
The 50X stock solution will be diluted 49:1 with water to make a 1X working solution. Final concentrations of the 1X solution are 40 mM Tris, 20 mM acetic acid, and 1 mM EDTA.

In this protocol you will find how to prepare complex media for growing Saccharomyces spp..

Reagents

Peptone
Yeast extract
Agar
Glucose
Demiwater

Protocol

Yeast Peptone Dextrose media is composed of YEP media and 2% glucose (from 40% stock). YEP is autoclaved separately and supplemented with Glucose after cooling.

Prepare 500 ml YEP media (Yeast Peptone):

Compound Name	Amount g/0.5L
Peptone	10
Yeast extract	5
Agar (only if making solid media for plates)	10

Mix components, add 475 ml ddH₂0 and autoclave. Components will dissolve during autoclaving. After YEP has cooled down, add 25 ml 40% glucose.

While working in the lab, we used different antibiotics for selection of our plasmids in E. coli. Here you can find a summary of the stock and working solutions.

Antibiotics:

Ampicillin
- Stock solution: 100 mg/mL
- Working solution: 100 µg/mL
- Dilution factor --> :1000
- Results in: 1 µl per ml media
Chloramphenicol
- Stock solution: or 25 mg/mL
- Working solution: 25 µg/mL
- Dilution factor --> :1000 (25mg/mL stock)
- Results in: 1 µl per ml media
Kanamycin
- Stock solution: 50 mg/mL
- Working solution: 50 µg/mL
- Dilution factor --> :1000
- Results in: 1 µl per ml media

In this protocol you will find how to prepare selective media for Saccharomyces spp.

Reagents

20x AA
Sterile water
2x SD
10x SD
40% Glucose
4% Agar
0.2% Uracil
1% Histidine
1% Tryptophan
1% Leucine

Preparation of 20x AA

Combine amino acid powders in a 1L beaker and add 500 ml ddH2O. Stir under light heating until fully dissolved. Sterile filter into a sterile 500 ml Schott bottle (autoclave the empty bottle before). *heat sensitive

Compound	Amount g/0.5L	Per liter	Final concentration
Adenine Sulfate	0.2	400 mg/L	20 mg
Arginine HCL	0.2	400 mg/L	20 mg
Aspartic acid*	1	2000 mg/L	100 mg
Glutamic acid	1	2000 mg/L	100 mg
Isoleucine	0.3	600 mg/L	30 mg
Lysine HCl	0.3	600 mg/L	30 mg
Methionine	0.2	400 mg/L	20 mg
Phenylalanine	0.5	1000 mg/L	50 mg
Serine	4	8000 mg/L	400 mg
Threonine*	2	2000 mg/L	200 mg
Tyrosine	0.3	600 mg/L	30 mg
Valine	1.5	3000 mg/L	150 mg

Preparation of 2x SD

SD = synthetic defined media w/o amino acids and carbon source. Add 6.7 g YNB (Yeast Nitrogen Base without amino acids with ammonium sulfate) per 500 ml ddH2O. Autoclave for 20 min at 120°C. Powder will fully dissolve during autoclaving.

Preparation of 10x SD

SD = synthetic defined media w/o amino acids and carbon source. Add 33.5 g YNB (Yeast Nitrogen Base without amino acids with ammonium sulfate) per 500 ml ddH2O. Autoclave for 20 min at 120°C. Powder will fully dissolve during autoclaving.

Preparation of 40% Glucose

40% (w/v) = 40 g per 100ml. 200 g per 500ml.

Note 1: Glucose will not dissolve easily. Heat up the water first and add glucose little by little while stirring.

Note 2: The Glucose powder takes a significant volume of 500 ml. The final volume is 500 ml including the volume of the glucose.

Weight 200 g glucose (hexose, dextrose) into a beaker. Use a second beaker and add 250 ml ddH2O. Heat water slightly under stirring. Add glucose little by little till fully dissolved. Add additional ddH₂O to the 500 ml final volume. Sterile filter into a sterile 500 ml Schott Bottle.

Preparation of 4% Agar

4% (w/v) = 4g per 100 ml.

Prepare maximal 300 ml per 500 ml Schott bottle. Add 12 g agar to 300 ml ddH₂O and autoclave. Agar will dissolve during autoclaving and solidify when cooled down. Either use directly after autoclaving to pour plates or heat and dissolve on a heat plate before using it.

Preparation of 0.2% Uracil

Add 0.4 g to 200 ml ddH2O and autoclave.

Preparation of 1% Histidine, 1% Tryptophan and 1% Leucine

Add 2 g to 200 ml ddH₂O and autoclave.

Preparation of Selective Media without uracil

To make 1 L of Selective Media (SM) without uracil, mix the following components

Component	V (mL)
10x SD (regular)	100
40% Glucose	50
20x AA	50
1% histidine	2
1% tryptophan	2
1% leucine	9
>demi H20	789

For making culture plates with SM-ura media, 100 ml 4% agar will be added and 100 ml less demi H₂O will be used.

In this protocol, you can find how the breakdown of starch by synthetic alpha-amylase can be tested using the starch plate halo test.

Materials

Starch plates
- Culture media + 1 % (potato) starch + 1,5 % agar
Iodine solution
- 1 g of iodine crystals + 2 g of potassium iodine in 300 mL of distilled water
Synthetic alpha-amylase
- 15 mg/L --> 2 mg of alpha-amylase in 133 mL SM-ura media

Protocol

Preparation of synthetic alpha-amylase dilutions

Prepare 7.5 mg/L alpha-amylase sample solution by mixing 500 µL of 15 mg/L alpha-amylase sample solution and 500 µL of SM-ura media.
Prepare 1.5 mg/L alpha-amylase sample solution by mixing 100 µL of 15 mg/L alpha-amylase sample solution and 900 µL of SM-ura media.
Prepare 0.75 mg/L alpha-amylase sample solution by mixing 500 µL of 1.5 mg/L alpha-amylase sample solution and 500 µL of SM-ura media.
Prepare 0.15 mg/L alpha-amylase sample solution by mixing 10 µL of 15 mg/L alpha-amylase sample solution and 990 µL of SM-ura media.
Prepare 0.075 mg/L alpha-amylase sample solution by mixing 400 µL of 0.15 mg/L alpha-amylase sample solution and 400 µL of SM-ura media.

Spotting of samples

Make 2 plates by spotting 5 µL of each sample and the stock solution in triplicate on a starch plate.
Incubate 1 plate 24h at 30ºC.
Incubate the other plate for 20 min at RT.

Halo test

Test the breakdown of starch by flooding the surface of the plates with a 1:1 iodine solution with MilliQ for 60 seconds. Discard any excess iodine from the plate.
Examine the clear zone around the culture spot. Measure the halo/take a picture from a solid point using the Fujifilm LAS-4000.

In this protocol you can find how the breakdown of starch by alpha-amylase can be tested using the starch plate halo test.

Materials

Starch plates
- Culture media + 1 % (potato) starch + 1,5 % agar
Iodine solution
- 1 g of iodine crystals + 2 g of potassium iodine in 300 mL of distilled water
ON culture of Saccharomyces spp.

Protocol

ON cultures

Culture 1 colony of each Saccharomyces spp. containing the cassette plasmid in 2 mL 2xSM-ura media overnight shaking at 30ºC.

Preparation of culture samples

Spin down all the samples at 3000 rpm in 1 min.
Dilute the samples to OD600 50

Spotting samples

Spot 5 µL of each sample in triplicate on a starch plate.
Incubate plates for 48h at 30ºC.

Halo test

Test the breakdown of starch by flooding the surface of the plates with an 1:1 iodine. solution with MilliQ for 60 seconds. Discard any excess iodine from the plate.
Examine the clear zone around the culture spot. Measure the halo/take a picture from a solid point using the Fujifilm LAS-4000.

Digestion with BsmBI was performed to assess the engineering success of all the constructs in the library.

Reagents

DNA template (SP₀₀₁-SP₀₆₄ + pRS₄₂₆-GFP)
10x Cut Smart Buffer 3.1 (NEB)
BsmBI
MilliQ water

Protocol

Prepare the digestion Eppendorf tubes as indicated in the table.

Components	Volume (μl)
10x Cut Smart buffer 3.1	2.5
BsmBI enzyme	0.5
DNA template (1 μg)	5
MQ	17
Total	25

Incubate the digestion for 1h at 37ºC.
Inactivate the enzyme by incubating the reaction at 80ºC for 20 minutes.

This protocol explains how to measure the alpha-amylase activity in the supernatant of overnight cultures using the alpha-amylase assay kit.

Reagents

Amylase Assay Buffer (Sigma-Aldrich, Amylase Activity Assay Kit)
Amylase Substrate Mix (Sigma-Aldrich, Amylase Activity Assay Kit)
Amylase Positive Control (Sigma-Aldrich, Amylase Activity Assay Kit)
Nitrophenol Standard (Sigma-Aldrich, Amylase Activity Assay Kit)
α-Amylase (Sigma-Aldrich, α-Amylase from Aspergillus oryzae)
- Molecular weight: 49 - 51 kDa
- Specific enzyme activity (pH 6.0, 25°C): ~30 U/mg
Milli-Q water
YPD media
96-well plate (3x)
200µL tube (48x)

Protocol

Preparation

Allow Amylase Assay Buffer and α-Amylase to warm up to RT.
Set the incubator to 25°C.
Transfer cultures into 200 µL tubes (homogenize by pipetting).

OD measurement

Add 190 µL of Milli-Q water to 48 wells.
Add 10 µL of one sample per well (homogenize by vortexing for 20 s).

Absorbance measurement

Add 0, 2, and 10 µL of Nitrophenol Standard in duplicates to 6 different wells on a 96 well plate. Add 50, 48, and 40 µL of Milli-Q water into the respective 6 wells on the 96 well plate, so the total volume of all wells equals 50µL.
Spin down the cultures in 200 µL tubes for 1 min at 8000 rpm, keep the supernatant as a sample.
Add 30 µL of each sample in triplicate to a well on a 96 well plate. Add 20 µL of Amylase Assay Buffer into the respective wells on the 96 well plate, so the total volume of both wells equals 50 µL.
Prepare Master Reaction Mix by mixing 7.1 mL (actually needed: 7.65 mL) of Amylase Assay Buffer and 7.1 mL (actually needed: 7.65 mL) of Amylase Substrate Mix.
Add 100 µL of Master Reaction Mix to each well.

Introduction

The change in absorbance over time in the supernatant from overnight liquid media cultures was measured to determine the alpha-amylase activity using the alpha-amylase assay kit. In this protocol you can find how to process the obtained data.

Materials

Absorbance measurements ($\vec{m}_i$, where $i$ is the sample number)
Absolute difference between the starting time and time of taking each of the measurements ($\vec{dt}$)
Average of duplicate blank standard ($\vec{s}_B$)
Average of duplicate 4 nmol standard ($\vec{s}_4$)
Average of duplicate 20 nmol standard ($\vec{s}_{20}$)
(optional) Average of duplicate 8 nmol standard ($\vec{s}_8$)
(optional) Average of duplicate 12 nmol standard ($\vec{s}_{12}$)
(optional) Average of duplicate 16 nmol standard ($\vec{s}_{16}$)

Protocol

Check whether $\vec{s}_B$, $\vec{s}_4$(, $\vec{s}_8$, $\vec{s}_{12}$, $\vec{s}_{16}$) and $\vec{s}_{20}$ remain constant over time. If all standards show some variation, correct $\vec{m}_i$ for all $i$ by subtracting with $\vec{s}_B$
Exclude all data points where $\vec{m}_i>\vec{s}_{20}$
Check whether a linear curve can be fitted to $\vec{m}_i$ such that $\vec{m}_i \approx a_i * \vec{dt} + b_i$ for all $i$
If the linear curve is a good fit, $a_i$ is the activity of sample $i$

Team:Groningen/Experiments

Introduction

Competent E. coli DH5α cells

Reagents

TYM medium

TfB I

TfB II

Protocol

E. coli transformation

Materials

Protocol

S. cerevisiae transformation

Reagents

PLATE Mixture

Other materials

Protocol

Notes

Part plasmid assembly - BsmBI Golden Gate

Reagents

Protocol

Cassette vector assembly - BsaI Golden Gate

Reagents

Protocol

PCR amplification of G-Blocks

Reagents

Protocol

Gel Electrophoresis

Reagents

Protocol

To make 1% agarose gel:

To run the gel electrophoresis:

50x TAE buffer

Reagents

Protocol

YPD media

Reagents

Protocol

Antibiotics concentrations

Antibiotics:

Selective Media

Reagents

Preparation of 20x AA

Preparation of 2x SD

Preparation of 10x SD

Preparation of 40% Glucose

Preparation of 4% Agar

Preparation of 0.2% Uracil

Preparation of 1% Histidine, 1% Tryptophan and 1% Leucine

Preparation of Selective Media without uracil

Starch test on synthetic alpha-amylase

Materials

Protocol

Preparation of synthetic alpha-amylase dilutions

Spotting of samples

Halo test

Alpha-amylase starch test on Saccharomyces spp.

Materials

Protocol

ON cultures

Preparation of culture samples

Spotting samples

Halo test

Digestion with BsmBI

Reagents

Protocol

Alpha-amylase assay kit

Reagents

Protocol

Preparation

OD measurement

Absorbance measurement

Obtaining alpha-amylase activity from absorbance measurements

Introduction

Materials

Protocol