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Experiments

Protocols of all Experiments we conducted

Drylab

Installing Gromacs (Linux OS or Linux subsystem):

Download g++ from cmake.org/download/ (cmake-version.tar.gz)
Download fftw from fftw.org/download.html (fftw-version.tar.gz)

In terminal:

Unpack cmake ( tar xzvf cmake-version.tar.gz)
Move to unpacked folder (cd cmake-version)
Run configuration (./configure)
Install cmake (make)
Install cmake (sudo make install)
Unpack fftw (tar xzvf fftw-version.tar.gz)
Move to unpacked directory (cd fftw-version)
Run configuration (./configure)
Install fftw (make)
Install fftw (sudo make install)
Install Gromacs (sudo apt-get install Gromacs)
Success!

Creating a membrane (All-Atom/Coarse-grained):

Membranes were created using the tool offered by CHARMM-Gui ^6, ^7, ⁸ .

Predicting protein structures:

To predict the structures of our grafted cyclotides we used AlphaFold Colab ⁴ .

Using Gromacs

As Gromacs or Molecular Dynamics simulation, in general, is a very complex and versatile topic, we are limiting the following section to what we learned about using Gromacs so far and how to set up a very basic system.

When starting with Molecular Dynamics, one should always consider that most simulations are very computationally intensive.
When the goal is to simulate a complex system (e.g., more than a protein in water) and a supercomputer is unavailable, one should consider using a so-called coarse-grained forcefield.
Forcefield is a data file used by Gromacs which contain numeric values required to compute interaction forces between different kind of atoms.
During our project we used 2 different kinds of forcefields to run our simulations:

All-Atom: a forcefield that computes interaction forced between each atom available in the simulation, very computationally intensive.
Coarse-grained: a forcefield that sums up groups of atoms into a single entity, describing it with the average chemical properties of the single atoms constituting it.

Thanks to coarse-grained forcefields (in our case Martini ⁵ ), we were able to run some simulations on our computers as well and achieved some good results, although, as might be clear already, the resulting simulations aren't as realistic as when using an all-atom forcefield.

File types:

To perform any kind of simulation, Gromacs uses the so-called .gro file format.
A .gro file contains the structural information of, e.g., a protein, molecule, or a composition of different molecular structures, and the size of the virtual box within this composition are placed.
In order to create such box for e.g. a protein, one can download the .pdb file of the protein and convert it into a .gro file (see Gromacs command pdb2gmx).

Apart from the virtual box containing all components, Gromacs requires (lots) of settings to be made, so it knows precisely how to calculate forces between atoms.
Those settings for the simulation are stored in .mdp files and contain information like which method shall be used to keep the temperature or pressure of the .gro file at the given values or how long the simulation is supposed to be.
As mentioned before, there are many settings to be made here, which reflects how many scenarios Gromacs can be applied.

Next-up are topology files (.top), which (in some way) contain a resume of a .gro file by listing its different molecules by name and in the same order.

The last file required to run a successful simulation is the index file (.ndx), which allows the user to sum up atoms of the .gro file into groups by their molecule number.
Note that this file only needs to be provided by the user if any non-standard groups are being used in the .mdp file (e.g., for coupling purposes), else it's automatically generated by Gromacs.

Running a simulation:

If the files described in the section above are at hand, it's possible to run the simulation!
For this purpose, a .tpr file is being generated by Gromacs, which sums up all settings and files used for the simulation program.

Before a so-called "production" (the simulation we want) can be run, a couple of short pre-simulations have to be made to have a physically correct virtual box (aka .gro file).
If we skip those pre-simulations, our system could contain overlapping atoms, or isn't set to a certain temperature or pressure!

Therefore the first simulation to be run is a so-called minimization, which is often also referred to as "relaxing" the system: atoms or molecules which, e.g., overlap or show other forms of inappropriate geometry are slightly moved to bring the total energy of the system under a certain threshold (previously defined in the .mdp file).

After lowering the total energy to a realistic value, it's time to bring the system to our desired temperature (which consists of the total kinetic energy of the system).
This simulation step is called NVT equilibration, where NVT stands for number, volume, and temperature, three characteristic values of the system which are tried to keep constant.
Temperature is introduced to the system by raising the kinetic energy of its particles.
These kinetic energies are adapted until the temperature plateaus (around) the value defined in the .mdp file.

After obtaining the desired temperature, we need to introduce a realistic pressure to our system.
This simulation is referred to as NPT equilibration, where, instead of volume, the pressure is tried to keep constant (therefore, the volume of the system must be modified).
The NPT simulation is considered successful once the density remains constant (note that the pressure can still fluctuate widely, therefore density is used as a review metric).

As mentioned before, pressure can still fluctuate wildly as instantaneous pressure is relatively meaningless and only makes sense as an average over time.

If the equilibration or minimization didn't yield the wanted result, it might be helpful to redo them, increasing their duration.

Now that our system has been successfully prepared, it's time for the production run (the simulation which gives us the result we want).
There are plenty of use cases for molecular dynamics simulations. Therefore, it is futile to describe a general approach in how a production run is supposed to be performed.
As a warning it may be said that although the minimization as well as the nvt and npt equilibration were successful, errors can still occur later on which may require extensive troubleshooting.

References

E. Lindahl, B. H. (2001). GROMACS 3.0: A package for molecular simulation and trajectory analysis. Journal of Molecular Modeling, 306-317.
H. Bekker, H. B. (1993). Gromacs: A parallel computer for molecular dynamics simulations. Physics computing, 252-256.
H.J.C. Berendsen, D. v. (2001). GROMACS: A message-passing parallel molecular dynamics implementation. Computer Physics Communications, 306-317.
Jumper, J. a. (2021). Highly accurate protein structure prediction with AlphaFold. Nature, 583-589.
Marrink, S. J. (2007). The MARTINI force field: coarse grained model for biomolecular simulations. The journal of physical chemistry B, 7812-7824.
Pin-Chia Hsu, B. M. (2017). CHARMM-GUI Martini Maker for modeling and simulation of complex bacterial membranes with lipopolysaccharides. Journal of Computational Chemistry, 2354-2363.
Sunhwan Jo, T. K. (2008). CHARMM-GUI: A Web-based Graphical User Interface for CHARMM. Journal of Computational Chemistry, 1859-1865.
Yifei Qi†, H. I. (2015). CHARMM-GUI Martini Maker for Coarse-Grained Simulations with the Martini Force Field. Journal of Chemical Theory and Computation, 4486-4494.

Wetlab

Agarose gel electrophoresis

Introduction

Separation of DNA fragments.

Materials

1x TAE-buffer
Agarose
nitrile gloves
loading dye: 6x purple loading dye from NEB
DNA ladder: GenLadder 1kbp (250 bp-10000 bp)
Diamond nucleic acid dye / Midori green
agarose gel chamber
power supply
UV-desk

Method

Weigh 2.5 g Agarose in a 250 ml screw-cap laboratory bottle.
Fill up to 250 ml with 1x TAE-buffer.
Heat up in microwave until agarose completely dissolves, take out of microwave and stir every 30 to 60 s. Store bottle in 60°C incubator or store at RT and heat up in microwave before using.
Pour agarose into gel tray. (If Midori green is used, add 1-2 µL when pouring agarose into the tray.) Let the gel solidify, covered with paper towel.
Put the gel into electrophoresis chamber, fill up with 1x TAE-buffer until gel is slightly covered.
Add 5 μl 6x loading dye to PCR-reaction, mix by pipetting.
Apply whole 25 μl of PCR-reaction supplemented with loading dye and 5 µl of DNA-ladder.
Run gel electrophoresis at 120 V for about 30 min (until dye front has run far enough).
If no Midori green is used, stain gel in Diamond nucleic acid dye (Promega) according to their protocol.
Analyze gel on UV-desk and take a picture.

Control digest

Introduction

To monitor cloning success.

Materials

Promega restriction enzymes: EcoRI (12 U/µl), BamHI (10 U/µl), HindIII (10 U/µl)
Promega 10x restriction buffers: buffer H (EcoRI), buffer E (BamHI, HindIII)
Promega acetylated BSA (10 mg/ml) as enzyme stabilizer
autoclaved ddH ₂ O
DNA sample
heat block

Method

In a sterile tube, assemble the following components in the order listed below:

Component	Volume (µl)
sterile ddH ₂ O	to 20
10x restriction buffer (Promega)	2
acetylated BSA (Promega, 10 mg/ml)	0.2
DNA sample	0.2 - 1.5 µg
restriction enzyme (Promega)	0.5
total	20

Mix gently by pipetting, close the tube and centrifuge for a few seconds in a microcentrifuge. Incubate at 37 °C for 1 - 4 hours.
Proceed with agarose gel analysis protocol

E. coli DH5α transformation

Introduction

Transformation of NEB 5-α chemically competent E. coli with cloning products; derived from NEB instructions .

Materials

C2987I NEB 5-α competent E. coli cells
DNA or cloning reaction
SOC medium (NEB)
LB selection plates
heat block
shaker/rotator

Method

Thaw a tube of NEB 5-α competent E. coli cells on ice. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
Add 1 - 5 µl sample containing 1 pg – 100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4 - 5 times to mix cells and DNA. Do not vortex.
Place the mixture on ice for 30 min.
Heat shock at 42 °C for 30 s.
Place on ice for 5 min.
Pipette 950 µl of SOC into the mixture.
Place at 37 °C for 60 min. Shake vigorously (250 rpm) or rotate.
Warm selection plates to 37°C.
Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
Spread 100 µl of each dilution onto a selection plate and incubate overnight at 37 °C.

GoldenGate BpiI (BbsI)

Introduction

Cloning level I and level III vectors with BpiI (BbsI).

Materials

Nuclease free H2O
T4 DNA ligase buffer (10x)
Plasmids/fragments
T4 DNA ligase (NEB)
BbsI-HF (NEB)
Thermocycler

Method

Set up 25 µl reaction in a PCR-tube by pipetting in the given order; pipet and store on ice:

Component	Amount
Nuclease free H2O	fill up to 25 µl
T4 DNA ligase buffer (10x)	2.5 µl
Plasmids/fragments	75 ng
T4 DNA ligase (NEB)	0.5 µl (200 U)
BbsI-HF (NEB)	1.5 µl (30 U)

Mix gently by pipetting up and down several times.

Run the following program in a thermocycler:

Temperature (°C)	Time (min)	Cycles
37	5	} 30x
16	5	} 30x
37	5	1x
60	5	1x

Transform 5 µl GoldenGate reaction into competent E. coli .

GoldenGate BsaI

Introduction

Cloning level II vectors with BsaI.

Materials

Nuclease free H2O
T4 DNA ligase buffer (10x)
Plasmids/fragments
GoldenGate assembly mix (NEB)
BbsI-HF (NEB)
Thermocycler

Method

Set up 20 µl reaction in a PCR-tube by pipetting in the given order; pipet and store on ice:

Component	Amount
Nuclease free H2O	fill up to 20 µl
T4 DNA ligase buffer (10x)	2 µl
Plasmids/fragments	75 ng
GoldenGate assembly mix (NEB)	0.5 µl (200 U)
BbsI-HF (NEB)	1 µl (20,000 U/ml)

Mix gently by pipetting up and down several times.

Run the following program in a thermocycler:

Temperature (°C)	Time (min)	Cycles
37	1	} 30x
16	1	} 30x
37	5	1x
60	5	1x

Transform 5 µl GoldenGate reaction into competent E. coli .

GoldenGate Esp3I

Introduction

Cloning cyclotides into level III vectors with Esp3I.

Materials

Nuclease free H2O
T4 DNA ligase buffer (10x)
Plasmids/fragments
T4 DNA ligase (NEB)
Esp3I (NEB)
Thermocycler

Method

Set up 25 µl reaction in a PCR-tube by pipetting in the given order; pipet and store on ice:

Component	Amount
Nuclease free H2O	fill up to 25 µl
T4 DNA ligase buffer (10x)	2,5 µl
Plasmids/fragments	75 ng
T4 DNA ligase (NEB)	0.5 µl (200 U)
Esp3I (NEB)	1,5 µl (15 U)

Mix gently by pipetting up and down several times.

Run the following program in a thermocycler:

Temperature (°C)	Time (min)	Cycles
37	5	} 30x
16	5	} 30x
37	5	1x
60	5	1x

Transform 5 µl GoldenGate reaction into competent E. coli .

Mini-/Midiprep

Introduction

Producing and multiplying plasmid DNA (e.g., after cloning) from E. coli .

Materials

LB agar plates
LB medium
appropriate antibiotics for selection
autoclaved glass test tubes
preparation kit (NEB Monarch® Plasmid DNA Miniprep Kit)
incubator
nanodrop

Method

Day 1:

Transform competent E. coli cells and plate them on LB agar plates supplemented with the appropriate antibiotics.
Incubate plates overnight at 37 °C

Day 2

Add 5 ml of LB medium with appropriate antibiotics into sterile glass test tubes.
Pick a colony from a plate with a sterile pipette tip or inoculation loop and stir in the 5 ml LB.
Grow the bacteria overnight in shaker at 37 °C.

Day 3

Grow bacteria for 8 h at 37 °C and shaking by inoculation of 50 ml LB with 50 µl preculture.
Spin down liquid cultures and proceed according to the preparation protocol by NEB.
Measure DNA concentration yielded from the preparation, using a nanodrop.

Sequencing

Introduction

To sequence plasmid or other DNA by Sanger sequencing.

Materials

Sequencing reaction
1.5 ml Eppendorf tubes
Labels

Method

Prepare plasmid according to protocol

Prepare sequencing reaction in 1,5 ml Eppendorf tubes: 15 µl, containing 40 - 100 ng/µl plasmid/DNA and 4 µM primer

Component	Amount
Plasmid/DNA	V (600-1500 ng)
20 µM primer	3 µl
Autoclaved ddH ₂ O	Fill up to 15 µl

Label the 1.5 ml Eppendorf tubes and send it to sequencing (Mycrosynth).

Inhibition zone assay

Introduction

To test antimicrobial properties of samples against E. coli and B. subtilis .

Materials

LB-agar plates
crude extract / purified protein solution
LB medium
E. coli cells
B. subtilis cells
glass tubes
filter paper discs

Method

Day 1:

Inoculate cells in 10 ml LB media overnight ( E. coli and B. subtilis ) in glass tube.

Day 2

Dilute overnight culture 1:10.
Inoculate new culture (OD ₆₀₀ = 0.1) and let it grow for 3 - 4 h until a OD ₆₀₀ = 0.8-1.0.
Dilute culture 1:50.
Prepare a series of dilutions of crude extract/protein solution: 1:10, 1:50, 1:50, 1:100, 1:500 and 1:1000 (200 μl each)
Spread 200 μl of 1:50 bacteria cultures onto LB-agar plates and let it dry.
Put small filter paper discs onto the LB-agar plates and add 20 μl of protein dilutions, each to one filter paper disc.
Incubate plates overnight at 37 °C.
Take photos of the petri dishes on the next day.

10x TAE-buffer

Introduction

Standard protocol for making 10x TAE-buffer.

Materials

Tris-base (M = 121.14 g/mol)
Glacial acetic acid (17,4 M)
EDTA (M = 372.23 g/mol)
ddH ₂ O

Method

Dilute 48.5 g Tris-base, 11.4 ml glacial acetic acid and 3.7 g EDTA in 800 ml ddH ₂ O.
Fill up with ddH ₂ O to 1 l.

1x Tricine running buffer

Introduction

Standard protocol for making 1x tricine running buffer.

Materials

100 mM Tris-base (M = 121.14 g/mol)
100 mM Tricine (M = 179.172 g/mol)
0.1% (w/v) SDS

Method

Weigh in the appropriate amount of chemicals.
Dissolve in ddH ₂ O and set pH to 8.3.
Fill up to desired volumed with ddH ₂ O.

Antibiotics

Introduction

Preparation and storage of antibiotics.

Materials

Gentamicin
Ampicillin
Kanamycin
Rifampicin
Streptomycin
Tetracycline

Method

Make solutions of 10 mg/ml gentamicin, 50 mg/ml ampicillin, 50 mg/ml kanamycin, 100 mg/ml rifampicin, 100 mg/ml streptomycin or 5 mg/ml tetracycline with ddH ₂ O.
Filter the antibiotic solutions with a 0.2 µM sterile filter under the hood and take 0.5 ml aliquots. Store them at - 20 °C.

Media

Introduction

Preparation of used media.

Materials

Müller-Hinton Broth (MH)
Brain Heart Infusion Broth (BHI)
LB broth
LB agar plates

Method

MH broth

Dissolve 21 g of MH broth in 1 l ddH ₂ O.
Autoclave the solution at 121 °C for 15 min.

MH broth

Dilute 10 ml of autoclaved MH broth with 40 ml of autoclaved water to obtain a 1/5 dilution.

MH broth

Dissolve 37 g of BHI broth in 1 l ddH ₂ O.
Autoclave the solution at 121 °C for 15 min.

MH broth

Dissolve 20 g of LB broth in 1 l ddH ₂ O.
Autoclave the solution at 121 °C for 15 min.

TE buffer

Introduction

Standard protocol for making TE-buffer.

Materials

Tris-base (M = 121.14 g/mol)
EDTA (M = 292.25 g/mol)
ddH ₂ O

Method

Dissolve 605.7 mg Tris and 14.6 mg EDTA in 400 ml ddH ₂ O.
Adjust the pH to 8.0 using a pH-meter
Fill up to 500 ml total volume with ddH ₂ O and autoclave.

Acetone protein precipitation

Introduction

To concentrate protein solutions, for SDS-PAGE or other methods not requiring properly folded proteins.

Materials

Acetone
Centrifuge

Method

Cool acetone to -20 °C.
Pipet protein sample solution into an Eppendorf tube and add four times the sample volume of acetone.
Vortex tube and incubate for 60 min at -20°C.
Centrifuge 10 min at 13.000-15.000 × g.
Decant and remove supernatant carefully.
Allow the acetone to evaporate from the uncapped tube at RT for 30 min.
Add buffer appropriate for the downstream process and vortex thoroughly to dissolve protein pellet.

Agrobacterium transformation

Introduction

Transformation of Agrobacterium tumefaciens with our 2in1 and 3in1 vectors for transient expression in tobacco.

Materials

A. tumefaciens strain AGL1
Antibiotics: 100 mg/ml Rif, 100 mg/ml Strep, 5 mg/ml Tet, 25 mg/ml Kan
Vector
LB medium
LB agar
Shaker
Incubator
Centrifuge

Method

Thaw Agrobacteria on ice
Add 3 µl plasmid DNA to 100 µl agrobacteria, mix by flicking the tube.
Incubate 5 min on ice.
Incubate 5 min in liquid nitrogen.
Thaw in hand and incubate 5 min at 37 °C.
Add 900 µl LB medium.
Incubate 3 h at 28 °C.
Centrifuge 2 min at 4000 rpm.
Remove 850 µl supernatant, resuspend pellet in remaining liquid.
Plate 100 µl of the suspension on LB agar plates containing 10 µg/ml Rif, 2.5 µg/ml Strep, 2.5 µg/ml Tet and 25 µg/ml Kan.
Incubate plates 2 days at 28 °C.

His-tag affinity purification

Introduction

How to purify His6-tagged peptide from tobacco crude extract. Protocol adapted from Promega and Potula et al., 2007 .

Materials

HisLink™ Protein Purification Resin (Promega)
Millipore filter (0.2 µm pore size)
Syringes
Column
Dialysis chamber, 2 kDa MWCO (ThermoFisher Slide-A-Lyzer™ Dialysis Cassettes)
Ultracentrifuge
Magnetic stirrer
Buffers for extraction, purification and dialysis (see table below)

Buffers	Extraction	Binding	Wash	Elution	Dialysis
Chemicals	150 mM NaCl	150 mM NaCl	150 mM NaCl	150 mM NaCl	150 mM NaCl
	3 mM KCl	3 mM KCl	3 mM KCl	3 mM KCl	3 mM KCl
	100 mM HEPES	100 mM HEPES	100 mM HEPES	100 mM HEPES	100 mM HEPES
	0.2-2 mM PMSF*	10 mM Imidazole	20 mM imidazole	500 mM Imidazole
	0.05% (v/v) Tween20*

*add freshly

Method

Cut off and weight infiltrated leaves.
Grind the leaves with liquid nitrogen using mortar and pestle in 200% per weight ice-cold extraction buffer.
Incubate 1 h on ice before proceeding. Invert every 5-10 min.
Centrifuge crude extract at 20.000 g for 30 min at 4 °C
Collect aliquot (200 μl) from the pellet.
Filter supernatant with Millipore filter, using pre-cleaned syringe.
Collect aliquot (200 μl) from the filtrate
Fill column with 1 ml HisLink resin and wait until resin has settled. Allow the column to drain, and equilibrate the resin with 5 column volumes (= 5 ml) of binding buffer, allowing the buffer to completely enter the resin bed.
Gently add the cleared, filtered lysate to the resin until the lysate has completely entered the column.
Collect aliquot from the flow-through.
Wash unbound proteins from the resin using 5 ml of binding buffer.
Collect aliquot from the flow-through.
Wash unbound proteins from the resin using 10 ml of wash buffer.
Collect aliquot from the wash.
Once the wash buffer has completely entered the resin bed, add elution buffer and start collecting 0.5 ml fractions.

Infiltration of tobacco using Agrobacterium tumefaciens

Introduction

Infect tobacco plants with genetically modified A. tumefaciens to produce our construct.

Materials

LB medium
Antibiotics (100 mg/ml Rif, 100 mg/ml Strep, 5 mg/ml Tet, 25 mg/ml Kan)
AS medium (see table below)
Tobacco plants
P19 bacteria solution
Syringe (1 ml)
Fluorescence microscope
Shaker

Chemicals	Concentration
MES-KOH buffer, pH 5.6	1 M
MgCl ₂	3 M
Acetosyringone in DMSO	150 M

Method

Inoculate 2 ml of LB medium with corresponding antibiotics with a single Agrobacterium colony.
Incubate over night at 28 °C while shaking.
Centrifuge bacterial culture for 15 min at 4000 rpm and discard the supernatant. P19 culture is centrifuged as well.
Resuspend the pellet in 2 ml of cold AS medium. Add AS medium until an OD ₆₀₀ of 0.8-1.0.
Combine 100 μl of the bacterial solution and 100 μl of P19 bacteria solution in a 2 ml Eppendorf tubes and fill up with AS medium to 2 ml.
Incubate on ice for at least 1 h.
Infiltrate 2 - 3 young tobacco leaves per plant with the bacterial solution.

A leaf gets agroinfiltrated — **Figure 1: Picture of how to perform the Agroinfiltration procedure.**
A 1 ml syringe is used to infiltrate the young tobacco plants from the underside of the leaves.

Peptide purification according to Poon et al. 2018

Introduction

Protocol for extraction and purification of peptides from Nicotiana benthamiana adapted from Poon et al. 2018.

Materials

Acetonitrile
Formic acid
Filter circles, 110 mm, 5-13 µm
Drying cabinet
Mortal and pestle
Magnetic stirrer
Round-bottomed flask
Rotation evaporator
RP-HPLC MS
MALDI-TOF MS

Method

Dry leaves overnight at 40 °C.
Grind leaves to fine powder with mortar and pestle.
Add 20 ml/g (dry weight) extraction buffer (50% acetonitrile - 1% formic acid in ddH ₂ O).
Incubate for 1 h at room temperature in a beaker glass on a magnetic stirrer.
Filter with filter paper to remove insoluble extract into round-bottomed flask.
Repeat incubation and filtering with insoluble extract with the same amount of extraction buffer.
Dry soluble fraction in round-bottomed flask with rotation evaporator.

Tricine SDS-PAGE

Introduction

SDS-PAGE (with tricin instead of glycine) to get a clear separation of small protein bands (1 to 5 kDa peptides); gel casting according to Schägger 2006.

Materials

10x anode buffer
10x cathode buffer
2x Tricine sample buffer
Prestained Protein Ladder - Extra broad molecular weight (5 - 245 kDa) (Abcam)
3x Gel buffer: 3 M Tris, 1 M HCl, pH 8.45, 0.3 % (w/v) SDS
Acrylamide AB-3 stock solution: 49.5 % T, 3 % C mixture
10 % APS
TEMED
Isopropanol
heat block
SDS gel chamber
Power source
Tray for staining the gel
Shaker
Scanner

Buffer	10x anode buffer pH 8.9	10x cathode buffer pH 8.25	2x Tricine sample buffer pH 6.8	3x Gel buffer pH 8.45
Chemicals	1 M Tris	1 M Tris	200 mM Tris/HCl	3 M Tris
	0.225 M HCl	1 M Tricine	2% (w/v) SDS	1 M HCl
		1 % SDS	40 % (v/v) glycerol	0.3 % (w/v) SDS
			0.04 % (w/v) Coomassie brilliant Blue R-250
			2 % (w/v) β-mercaptoethanol

16 % Separation gel	Component	Amount for 30 ml
Chemicals	AB-3	10 ml
	3x Gel buffer	10 ml
	ddH ₂ O	10 ml
	10 % APS*	100 μl
	TEMED*	10 μl

*add latest

4 % Stacking gel	Component	Amount for 12 ml
Chemicals	AB-3	1 ml
	3x gel buffer	3 ml
	ddH ₂ O	8 ml
	10 % APS*	90 μl
	TEMED*	9 μl

*add latest

Method

Pour 16 % separation gel.
Cover separation gel with isopropanol until it is fully polymerized.
Pour 4 % stacking gel on top.
Add comb and let gel polymerize.
Mix 10 µl sample with 10 µl 2 x sample buffer by pipetting (For flowthroughs and crude extracts dilute 1:10 to prevent gel overload. For samples with known protein concentration use 15 - 20 µg protein).
Heat samples at 95 °C for 10 min.
Spin down samples shortly in centrifuge.
Apply 15 µl of each sample onto the gel; apply 10 µl protein ladder.
Run SDS-PAGE for about 100 min at 100 V.
Stain gel for 20 min to 1 h in Coomassie blue staining solution, either PageBlue™ Protein Staining Solution (Thermo Fisher Scientific) or Der Blaue Jonas ultrafast protein stain (GRP), shaking at RT.
Collect staining solution, wash gel in ddH ₂ O for 10 min.
Take gel pictures using the scanner.

Western blot

Introduction

To specifically detect proteins separated by SDS-PAGE using antibodies.

Materials

1x Blotting buffer
TBS-T
Milk-powder
100 % Methanol (can be reused)
1x PVDF membrane (Immobilion)
4x Whatman-paper, 8.6 x 6.2 cm
Mouse anti-His antibody
Anti-mouse HRP-coupled antibody
Luminescence reagents (Roche Lumi-Light Western Blotting substrate)
Semi-dry blotter
Rotator

Buffer	1x blotting buffer	TBS-T pH 7.4
Chemicals	25 mM Tris	150 mM NaCl
	19.2 mM glycerol	0.1 % Tween20
	20 % (v/v) MeOH	50 mM Tris/HCl

Method

Perform SDS-PAGE
Activation of the PVDF-membrane in 100 % MeOH; afterwards equilibration in blotting buffer.
Equilibration of Whatman-papers (4 X) and SDS-gel in blotting buffer.
Assemble blotting sandwich:

Assemble blotting sandwich — **Figure 2: Set-up of the blotting sandwich for western blotting.** We used a Semi-Dry blotting technique. Therefore, the PVDF membrane was activated in methanol and two layers of Whatman paper, preequilibrated in blotting buffer, were placed above and under gel or membrane, respectively.

Run blot at 20 V (1000 mA) for 30 min.
Block the membrane for 1-2 h at RT ( or O.N. at 4 °C) on a rotator with 5% milk-powder/TBS-T
Wash 3 times (each 10 - 15 min) with TBS-T.
Transfer membrane in a clean 50 ml tube.
Incubate with mouse anti-His antibody (diluted 1:1000 (10 µl AB in 10 ml TBS-T or 5% milk/TBS-T) rotating O.N. at 4 °C ( or for 1 h rotating at RT).
Wash 3 times (each 10 - 15 min) with TBS-T.
Incubate with anti-mouse HRP-coupled antibody (diluted 1:5000 (2 µl in 10 ml TBS-T); wrap the 50 ml tube in aluminum foil to protect the HRP-coupled antibody from light damage) for 1 h rotating at RT.
Wash 3 times (each 10 - 15 min) with TBS-T.
Mix detecting reagent A and B in a 1:1 ratio (300 µl A and 300 µl B, for 6.2 x 8.6 cm membrane). Mix the reagents shortly before use in an Eppendorf tube wrapped in aluminum foil and store it on ice.
Put the membrane on a plastic foil, distribute detection reagent on the membrane, cover the membrane with another plastic foil and remove air bubbles. Incubate for 5 min at room temperature (covered from light).
Detect luminescence with increasing exposure time step-wise (e. g. 5 s, 1 min, 5 min, 10 min). Take a picture of the ladder with an exposure time of 0.1 s.

Activity Testing – microdilution assay
(Koehbach et al. 2021 / Wiegand et al. 2008)

Introduction

Purified construct or crude extract will be used to test the antimicrobial activity. We will use several different methods for testing since the antimicrobial effects of AMPs can be hidden by different factors of an assay. To overcome this problem, we will do many different assays.

Materials

96-well plates (sterile, polypropylene, U-bottom (Nunclon Delta polystyrene))
Mueller-Hinton I Broth (MH)
Brain Heart Infusion Broth (BHI)
E. coli K12
Bacillus subtilis

Method

Assay with MH Broth

Twofold serial dilution of the antibiotics and peptides or of crude extracts using the respective media (MH, 1/5 MH, BHI).
For sterility control use media without bacterial solution.
For growth control use media with bacteria but without peptide/antibiotics.
Prepare 10 ml of a 1x10 ⁶ CFU/ml (bacterial cultures should be in log-phase):
For this, take 3 - 5 colonies of a plate and resuspend in 500 µl saline (0.9 % NaCl).
Pipette 100 µl of this bacterial suspension into a cuvette and add 900 µl saline (1:10 dilution). Measure OD ₆₀₀ for CFU/ml determination.
Dilute cells depending on needed concentration (OD ₆₀₀ = 0.1 equals E. coli BW25113: 1.4x10 ⁸ CFU/ml; B. subtilis 168: 2x10 ⁷ CFU/ml.)
Add 50µL bacteria suspension with the needed concentration to the wells.
Incubate plates at 37 °C O.N.
Measure OD ₆₀₀ .

SDS-extraction

Introduction

We could not extract our peptide with the former extraction buffer (100 mM HEPES, pH 7.5, 150 mM NaCl, 3 mM KCl), but with Lämmli buffer containing SDS. Therefore, extraction might be successful with a new extraction buffer containing SDS.

Materials

extraction buffer
precipitation buffer
Mortar and pestle
centrifuge
dialysis chamber
exicator

Buffer	extraction buffer pH 8.8	Precipitation buffer
Chemicals	125 mM Tris/HCl	90 % (v/v) acetone
	10 % (v/v) glycerol	5 % (v/v) triethylamine
	1 % (w/v) SDS	5 % (v/v) acetic acid

Method

Cut off and weight infiltrated leaves.
Grind the leaves with liquid nitrogen using mortar and pestle.
Add 200 % (v/w) extraction buffer
Centrifuge shortly to move down plant remnants, mix briefly afterwards.
Incubate on ice for 1 h.
Centrifuge 2 x 10 min with 4200 rpm, at 4 °C.
Take supernatant into dialysis chamber, dialysis at 4 °C O.N.
Take sample out of the dialysis chambers.
Add 19 x volume of precipitation buffer, mix gently, incubate on ice for 1 h.
Centrifuge 10 min at 4 °C with 4200 rpm, discard supernatant.
Wash pellet 2 x with 1 ml precipitation buffer.
Wash pellet 2 x with 1 ml acetone.
Dry pellet in the exicator.
Dissolve pellet in destination buffer.

Mass spectrometry of whole proteins eluted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels

Introduction

Protein bands are excised from SDS-PAGE gels, destained, and extracted for subsequent mass spectrometric analysis of whole proteins.

Materials

Acetonitrile
Ammonium bicarbonate
Trifluoroacetic acid
HEPES extraction buffer for re-solubilisation (10 mM HEPES, 15 mM NaCl, 0.3 mM KCl, pH 7.5)

Buffer	destaining buffer	extraction buffer
Chemicals	50 % (v/v) acetonitrile	50 % (v/v) acetonitrile
	50 % (v/v) ddH20	45 % (v/v) ddH20
	(+ 100 mM (NH4)HCO3)	5 % (v/v) TFA

Method

The respective protein bands were excised from the Coomassie-stained SDS-PAGE gels with a scalpel and transferred into 1.5 ml tubes.
Destaining of the gel slices was performed two times in 1 ml destaining buffer 1 (AcN:ddH ₂ O 1:1) and two times in 1 ml destaining buffer 2 (buffer 1 + 100 mM (NH ₄ )HCO ₃ ). The tubes were placed on a heat block (40 °C) for about 15 minutes and rigorously vortexed in between until the blue color faded.
Afterwards the buffer was removed and the gel slices were crushed using tweezers. The crushed gel slices were then incubated in 1 ml extraction buffer at 30 °C for about two hours on a rotator.
Thereafter the solvent was evaporated on a vacuum concentrator O.N. and the extract was re-solubilized in 10 µl of 1:10 diluted HEPES extraction buffer.
After the first measurements the samples were additionally concentrated and desalted using C18 resin.
The samples were analyzed via MALDI-TOF MS (Bruker) and LC-MS.

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About Us

We are the iGEM Team Tuebingen, a group of motivated students who are working on creating a fast screening platform for stabilized peptides. We are aiming to provide a system that gives everyone the ability to stabilize peptides such as antimicrobial peptides to create better medical agents.