Experiments

Electrophoretic Mobility Shift Assay (EMSA)

Electrophoretic Mobility Shift Assay (EMSA) is used to study protein-nucleic acid interactions by analyzing the binding of proteins to specific DNA sequences. It detects the formation of protein-nucleic acid complexes based on their slower migration through a polyacrylamide gel compared to free nucleic acids. Based on the results of an EMSA, quantitative values like the apparent dissociation constant K_d can be calculated.

Preparation of Samples

• DNA fragments can be ordered as oligos or extracted from cell lysate. In general, DNA fragments 20 - 40 nt in length are used in concentrations under the dissociation constant of the protein-nucleic acid complex.
• Use purified proteins in appropriate buffers under physiological conditions.
• The protein concentration must be optimized to detect specific binding without overwhelming the system with non-specific interactions. Protein concentration should be titrated from a concentration that lies above the dissociation constant to a concentration that lies below the dissociation constant of the protein-nucleic acid complex.
• Equilibrate samples for 30 min at 4 - 20 °C depending on the protein-nucleic acid complex (Note: Non-protein components should be pre-mixed and brought to temperature before adding proteins).

Native-PAGE Gel Electrophoresis

1. Pre-run native PAGE gel in running buffer at 200 - 300 V for at least 30 min.
2. Load samples in 1x Native PAGE loading dye (should contain 2 - 5% glycerol).
3. Run gel at 100 V for approximately 30 min.
4. To detect DNA, incubate the gel in running buffer with SYBR Safe (1:10 000 diluted) for 30 min. Image gel with transilluminator.
5. Wash gel twice with ddH₂O.
6. To detect proteins, incubate the gel in Coomassie staining solution for 30 min.
7. Destain with Coomassie destain solution.

FRET Bacterial Growth Assay

Förster Resonance Energy Transfer (FRET) is a distance-dependent interaction phenomenon between two fluorescent molecules, where energy is transferred from an excited donor fluorophore to a nearby acceptor fluorophore without emission of a photon. The donor is excited by absorbing light at a specific wavelength, and if the acceptor is within close proximity (1-10 nm), the energy is transferred, leading to emission from the acceptor. In FRET experiments, the key measurement is the emission intensity of the acceptor, while the donor's fluorescence is reduced. This process provides valuable information about molecular distances and interactions.

How to measure FRET with a bacterial culture:

1. Prepare an overnight culture by inoculating 4 mL of LB medium supplemented with the appropriate antibiotics with a single bacterial colony from a LB agar plate. Incubate at 37 °C overnight while shaking.
2. Preparation of pre-culture:
• Set up a pre-culture by inoculating 10 mL of LB medium supplemented with the appropriate antibiotics with 100 µL of the overnight culture.
• Incubate pre-culture at 37 °C while shaking until the culture reaches an OD₆₀₀ of 0.1 (dilute with LB medium supplemented with the appropriate antibiotics if OD₆₀₀ is too high).
3. Preparation of 96-well plate:
• Pipette 200 µL of LB medium containing 1.6 mM IPTG and the appropriate antibiotics into the first row of a 96-well plate using a multichannel pipette.
• Pipette 100 µL LB medium in the remaining wells.
• Perform a 1:2 dilution series from row A to G:
• Pipette 100 µL from the wells of the first row into the second row and mix by pipetting up and down.
• Repeat for the remaining rows but leave out row H.
4. Pipette 100 µL of the bacterial culture in each well (leave out one column and add 100 µL of LB medium instead; avoid bubbles while pipetting).
5. Measure OD₆₀₀, fluorescence intensity, and FRET fluorescence intensity with a TECAN plate reader.
• Measure the fluorescence intensity of both donor and acceptor fluorophores separately. And for the FRET interaction.
• Calculate the FRET efficiency as a ratio of acceptor emission to donor emission, correcting for background fluorescence.

Dual-Luciferase Assay

The Dual-Luciferase Assay utilises two types of luciferase: Renilla and Firefly. Renilla luciferase serves as an internal control and oxidises the substrate Coelenterazine, emitting light at approximately 480 nm. Firefly luciferase, on the other hand, acts as a reporter of gene expression by catalysing the oxidation of luciferin, which results in light emission at a wavelength of around 550-570 nm. This assay requires two key reagents from the Dual-Glow Luciferase Assay system from Promega: LAR II, which contains luciferin, and the STOP & Glo reagent, which halts the firefly luciferase reaction while introducing the Renilla substrate, Coelenterazine.

Materials

• White 96-well plate
• Dual-Luciferase® Reporter Assay System
• MiliQ water
• 70% Ethanol

Preparation of LARII reagent

• Thaw buffer in water
• Use a serological pipette, take up 5 mL LARII buffer (Green cap) and resuspend LARII Substrate (powder in an amber glass bottle), transfer to a brown 15mL falcon tube
• Take up another 5mL LARII buffer and repeat
• Wrap falcon in aluminium foil
• Freeze at -80°C or use directly

Preparation of STOP&Glow reagent

• Thaw buffer in water
• Transfer 10mL STOP&Glow buffer to a brown 15 mL Falcon tube
• Add 200 µL Stop&Glow reagent (1.5mL reaction tube with blue cap)
• Wrap falcon in aluminium foil
• Freeze at -80°C or use directly (Protect from Light at all times)

Execution of the measurement

Day 1

• Seed HEK293T cells into a transparent 96-well plate with a density of 12.500 cells/well

Day 2

• Transfect cells with the respective transfection plan & incubate ~48h at 37°C & 5% CO2

Day 4

• Before lysis, take out LAR II & STOP&Glow Buffers from -80 °C and thaw in water (35 µL/well of each reagent + 2 mL dead volume for priming)
• Prepare passive Lysis Buffer (needs 30 µL/well, dilute from 5x to 1x)
• Discard media from the 96-well plate by a quick swing over the S1-bin (not too hard otherwise HEK cells will be flung out) and then lyse with 30 µL/well passive Lysis Buffer for 30 min at RT & 500 rpm (tape to a thermomixer and set to 25 °C)
• While Lysis takes place, prepare TeCan Reader by washing the injectors twice with water, then twice with air
• When thawed, place the left injector into 15 mL Falcon of LAR II and the right injector into 15 mL Falcon of STOP&Glow
• Start Priming
• Measure with a protocol first dispensing 30 µL of LARII integrating for 8 seconds followed by 30 µL of STOP & Glow, again integrating for 8 seconds
• After Lysis, either transfer 10 µL/well to a new, white 96-well plate or seal with aluminium foil and store at -20°C
• Place the white 96-well plate into the TeCan reader and start the measurement
• After the measurement, remove the plate, backflush the reagents and wash the injectors three times with water, twice with 70% ethanol, then twice with air

RT-qPCR

RT-qPCR (Reverse Transcription Quantitative Polymerase Chain Reaction) is a powerful technique used to quantify gene expression levels in various biological samples. It combines reverse transcription of RNA into complementary DNA (cDNA) with PCR, enabling the precise quantification of specific RNA molecules.

1. RNA isolation is performed from tissue or cells using either the miRNeasy Tissue/Cells Advanced Mini Kit or the RNeasy Mini Kit, following the manufacturer’s instructions.
2. Isolated RNA is reverse transcribed into cDNA using the iScript Advanced cDNA Synthesis Kit for RT-qPCR, according to the manufacturer’s protocol.
3. Preparation of cDNA Synthesis Reaction Mix:
Table 18: Typical composition of a cDNA Synthesis Reaction Mix.

Component	Volume
5x iScript Advanced Reaction Mix	4 μL
iScript Advanced Reverse Transcriptase	1 μL
Isolated RNA (100 fg – 7.5 µg)	variable
Nuclease-free water	to 20 μL

4. Incubate the reaction mix in a thermocycler under the following conditions:
• Reverse transcription: 20 min at 46 °C
• RT inactivation: 1 min at 95 °C
• (Note: Alternatively, use 30 min at 42°C and 1 min at 95°C.)
5. After cDNA synthesis, dilute the cDNA based on RNA input:
   • For high RNA input (1.0 – 7.5 µg), dilute cDNA at least 10-fold
   • For low RNA input (< 1 µg), use cDNA directly in qPCR
6. Preparation of RT-qPCR Reaction Mix (Total Volume: 15 μL):
Table 19: Typical composition of a RT-qPCR Reaction Mix .

Component	Volume
iQ SybrGreen Master Mix	7.5 μL
Forward primer (300 nM)	0.5 μL
Reverse primer (300 nM)	0.5 μL
Diluted cDNA	6.5 μL

7. Mix gently and spin down.
8. Pipette samples onto plate and seal wells with optically transparent film.
9. Run the qPCR using the following program:
Table 20: Thermocyler program for a qPCR.

Step	Temperature	Time	Return to step	# Passes total
Polymerase Activation and DNA Denaturation	95°C	30 s		1
Denaturation	98°C	5 –15 s		35 - 40
Annealing/Extension and Plate Read	55 – 60 °C	15 –30 s	2	35 - 40
Melt-Curve Analysis	65 – 95 °C with 0.5 °C increments	2 – 5 s per step		1

10. (Note: Shorter annealing/extension times (1 – 10 s) can be used for amplicons <100 bp.
    Longer annealing/extension times (30 – 60 s) can be used for amplicons >250 bp.)

T7 Endonuclease I Assay

The T7 Endonuclease I (T7E1) assay is a widely used method to detect CRISPR-Cas-induced mutations, such as insertions or deletions (indels), at targeted genomic loci. After CRISPR editing, T7E1 recognizes and cleaves mismatched DNA in heteroduplexes formed when edited and unedited DNA strands are hybridised. The resulting DNA fragments can then be visualised using agarose gel electrophoresis. This assay provides a quick and efficient way to assess the efficiency of CRISPR-mediated genome editing by detecting the presence of indels at the target site.

Materials

• DirectPCR-Cell lysis reagent
• Proteinase K
• NEBuffer 2
• T7 Endonuclease I

Cell Processing

Day 1: Cell Seeding

• Seed 12,500 cells per well into a 96-well plate and incubate overnight at the appropriate conditions for cell growth.

Day 2: Transfection

• Transfect cells by adding 200 ng of plasmid DNA and Lipofectamine 2000 transfection reagent to each well, following the manufacturer’s protocol for optimal transfection efficiency.

Day 5: Cell Lysis and DNA Amplification

• Lysate preparation: Add 70 µL of sterile H₂O, 70 µL DirectPCR Cell Lysis Reagent, and 1.4 µL Proteinase K to each well.
• Incubate cells for 6 hours at 55 °C with gentle shaking (120 rpm).
• Inactivate enzymes by heating at 85 °C for 45 minutes.
• Amplify the targeted gene regions by adding 3 µL of the lysate to a 25 µL PCR reaction using Q5 High-Fidelity DNA Polymerase and specific primers.

Post-PCR Processing

• Pour 2% agarose gel in 1xTBE.
• Transfer 5 µL of the PCR product into a new tube with 2 µL NEB Buffer 2 and 13 µL nuclease-free water.
• Subject the sample to a temperature gradient: reduce from 95 °C to 85 °C at a rate of 2 °C/s, then from 85 °C to 25 °C at 0.1 °C/s.
• Keep samples on ice from now on!

T7 Endonuclease I Digestion

• Add 0.5 µL of T7 Endonuclease I to the reaction and incubate at 37 °C for 15 minutes to cleave mismatched DNA.

Gel Electrophoresis

• Immediately add 5 µL of purple loading dye (no SDS) with 1% GelRed to the digested samples.
• Run the samples on the already prepared gel to visualise the cleaved DNA fragments.
• Analyse the gel images using ImageJ to quantify the extent of cleavage.

Inter-Bacterial Conjugation Assay

Solid Media Conjugation Assay

• Transform corresponding plasmids into chemically competent E. coli strains.
• Plate the transformed cells on LB agar plates containing the appropriate antibiotics.
• Pick single colonies and set up 5 mL overnight cultures in LB medium with antibiotics.
• Reserve 500 µL of the liquid cultures as glycerol stocks, store at -80°C for future use.
• Centrifuge the remaining cultures at 11,000 rpm for 1 min in separate 1.5 mL tubes.
• Resuspend the pellets in 100 µL of 10 mM magnesium sulfate solution.
• Adjust the OD₆₀₀ of each suspension to 10 using the magnesium sulfate solution.
• Prepare the experimental groups by mixing 100 µL of recipient and donor cell suspensions.
• Add 1 mL of magnesium sulfate solution to each tube, vortex, and centrifuge at 11,000 rpm for 1 min.
• Remove the supernatant and resuspend the cell pellets in 10 µL of magnesium sulfate solution.
• Pipette 10 µL of the mixture onto an antibiotic-free LB agar plate and let air dry for 10 min.
• Incubate the plates at 37°C and allow conjugation to proceed for 18 hours.
• Collect bacterial patches using sterile inoculation loops and resuspend in 1 mL of magnesium sulfate solution.
• Adjust the OD₆₀₀ of all suspensions to 2.4.
• Prepare serial dilutions from 10^-2 to 10^-9 using the OD-adjusted suspensions.
• Plate the dilutions on two types of LB agar plates:
  • One selecting for recipients.
  • One selecting for transconjugants.
• Incubate the plates at 37°C overnight.
• Calculate conjugation efficiency by dividing the number of transconjugant colonies by the number of recipient colonies at a particular dilution.

Liquid Media Conjugation Assay

• Use the same E. coli clones as in the solid media conjugation assay.
• Follow the first five steps of the solid media conjugation assay.
• Resuspend the pellets in 1 mL of antibiotic-free LB medium.
• Adjust the OD₆₀₀ of each suspension to 10.
• Mix 120 µL of the OD-adjusted recipient and donor cultures for each experiment group.
• Centrifuge the mixtures for 1 min at 11,000 rpm.
• Resuspend the pellets in 1.2 mL of LB medium.
• Pipette 1 mL of each suspension into a well of a 12-well plate.
• Incubate the plate at 37°C for and allow conjugation to proceed for 18 hours without shaking.
• After incubation, remove the bacterial suspensions from the wells and adjust their OD₆₀₀ to 2.4.
• Prepare serial dilutions as for solid media conjugation and plate on selective agar plates.
• Incubate the plates at 37°C overnight.
• Calculate conjugation efficiency by dividing the number of transconjugant colonies by the number of recipient colonies at a particular dilution.

Co-Immunoprecipitation (Co-IP) Assay

Transfection and Lysis of HEK293T Cells

Cell Seeding

• Seed HEK293T cells in 6-well plates at a density of 300,000 cells per well.
• Grow them in 2 ml of DMEM supplemented with 10% FCS, 2 mM L-glutamine, and 1% penicillin/streptomycin.
• Incubate for 24 hours at 37°C in a humidified incubator (5% CO₂).

Transfection

• After 24 hours, transfect each well with 1 µg of EGFR-GFP plasmid (Addgene #32751) using Lipofectamine 2000 (Thermo Fisher Scientific), as per the transfection protocol.

Lysis

• 48 hours post-transfection, lyse cells using an NP 40-based lysis buffer (50 mM HEPES-NaOH (pH 8), 100 mM NaCl, 1 mM EGTA, 0.5% IGEPAL CA-630, 2.5 mM MgCl₂, 1 mM DTT, 10% glycerol) supplemented with protease inhibitors.
• Add 200 µl of ice-cold lysis buffer per well and incubate on ice for 15 minutes.
• Scrape cells, transfer to 1.5 ml tubes, and sonicate (30% power, 15 mins on ice).
• Centrifuge at 20,800g for 30 minutes at 4°C.
• Measure protein concentration (for example using the DC Protein Assay Kit (Bio-RAD)).
• Store the supernatant at -20°C for future use.

Induction of Protein Expression and Lysis of E. coli

Transformation and Culture

• Transform E. coli 10-beta cells separately with plasmids: pNeae2_7D12 and pNeae2.
• Plate cells on LB agar plates with chloramphenicol (25 µg/ml).
• Inoculate 5 ml overnight cultures in LB-chloramphenicol supplemented with 1% glucose for lac repression.

Induction

• Dilute overnight cultures 1:100 into 250 ml LB-chloramphenicol.
• Grow until OD₆₀₀ reaches 0.4-0.6, then induce with 50 µM IPTG.
• Incubate for 6-8 hours at 37°C.

Lysis

• Harvest bacteria by centrifugation (5150 rpm, 20 mins, 4°C).
• Resuspend bacterial pellets in NP 40-based lysis buffer with protease inhibitors (2 ml per gram of pellet).
• Incubate with lysozyme (1 mg/ml) for 35 minutes at 4°C.
• Sonicate the lysates (40% power, 15 mins on ice).
• Add DNase I (25 U/ml) and incubate for 30 minutes at 4°C.
• Centrifuge at 5150 rpm for 30 minutes at 4°C.
• Measure protein concentration (for example using the DC Protein Assay Kit (Bio-RAD)).
• Store the supernatant at -20°C for future use.

Co-immunoprecipitation (CoIP)

Preparation of Anti-myc Beads

• Resuspend agarose anti-myc beads (Proteintech) and transfer 25 µl bead slurry into five 1.5 ml tubes.
• Equilibrate beads with 500 µl of ice-cold dilution buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, pH adjusted at 4°C).
• Centrifuge at 4500 rpm for 5 minutes at 4°C, discard the supernatant.

Binding and Washing

• Dilute HEK293T and E. coli lysates (400 µg total protein) in 500 µl of ice-cold dilution buffer.
• Add lysates to equilibrated beads and rotate end-over-end for 1 hour at 4°C.
• Centrifuge at 4500 rpm for 5 minutes at 4°C, discard the supernatant.
• Wash beads with 500 µl of cold wash buffer (10 mM Tris/Cl pH 7.5, 150 mM NaCl, 0.05% IGEPAL CA-630, 0.5 mM EDTA) three times.

Elution

• Elute bound proteins by resuspending the beads in 80 µl of 2x SDS sample buffer (Laemmli) and boiling for 5 minutes at 95°C.
• Centrifuge at 4500 rpm for 2 minutes, collect the supernatant.

Analysis

• Perform SDS-PAGE (using 4-15% precast gradient gel, Bio-RAD).
• Analyze the resolved proteins by performing a Western Blot using primary antibodies against GFP (rabbit anti-GFP, 1:1000) and myc (mouse anti-myc, 1:1000).
• Use fluorophore-coupled secondary antibodies for detection (goat anti-rabbit DyLight 800 4X PEG and goat anti-mouse Alexa Fluor™ 680).

Experimental Groups

• Sample 1: Negative control, testing anti-myc bead cross-reaction.
• Samples 2-3: Verify presence of myc epitopes on proteins of interest.
• Samples 4-5: Co-IP experiments for nanobody-antigen interaction (Sample 4 as control, Sample 5 as test group).

Adhesion Assay

Preparation of Mammalian Cells

• Coat coverslips in two 12-well plates with poly-D-lysine as follows:
• Prepare 1:10 poly-D-lysine stock solution.
• Place one coverslip in each well and add 0.5 ml of poly-D-lysine stock solution, incubate for 15 minutes at RT.
• Wash twice with 1 ml PBS.
• Let plates dry by leaving them open in the sterile hood for 2 hours.
• Close plates and keep them wrapped in parafilm at 4°C for up to two weeks.
• Seed HeLa and HEK293T cells in 12-well plates on coverslips (10⁵ cells/well).
• Let cells grow in the humid CO₂ incubator for 24h.

Preparation of Fluorescent E. coli

• Set up overnight cultures of the various bacterial strains in LB-media with appropriate antibiotics:
  • E. coli NEB 10-beta with pHelper_RP4 (negative control without adhesin).
  • E. coli NEB 10-beta with pHelper_RP4 + pNeae2 (negative control without nanobody);
  • E. coli NEB 10-beta with pHelper_RP4 + pNeae2_7D12 (test group);
• Bring to an OD₆₀₀ of 0.1 (set up 2 tubes for test group and 2 tubes for each control), let grow until an OD₆₀₀ of 0.5 and inoculate with 50 µM IPTG to induce adhesin expression.
• Incubate for 2 hours at 37°C in a shaker.
• Harvest bacteria by centrifugation (4000xg, 3 min) and bring once again to an OD₆₀₀ of 0.1, inoculate with 10 µM IPTG and let grow until OD₆₀₀ of 0.5 (measure tightly to catch the beginning of exponential phase).
• Add RADA (stock in DMSO, final concentration in culture = 0.5 mM) in one tube of test group and negative control and incubate at 37°C for 2 hours (culture volume = 3 ml) [keep one culture each without RADA].
• Harvest bacteria from the tubes by centrifugation (4000xg, 3 min) and wash twice with PBS
• Check fluorescence with plate reader and compare to cells grown in media without FDAA. Excitation/emission wavelengths of RADA: approx. 554/580 nm.
• Pellet bacteria (only the samples labelled with RADA) and keep them on ice until resuspension.

Infection with E. coli Expressing Adhesin

• Remove media from the 12-well flasks containing seeded HeLa and HEK293T cells, add DMEM without FCS and antibiotics (2 ml per well).
• Resuspend bacteria in PBS or DMEM to reach an OD of 0.1 (roughly 3x10⁷ cfu/ml).
• Perform outside sterile hood! Add 1 ml of fluorescently labelled bacteria per well (to reach MOI 300:1), 1 hour infection time at 37°C.

Fixing of Cells

• Aspirate media and wash the coverslips five times with 1 ml PBS (RT).
• Fix coverslips with 0.5 ml paraformaldehyde 4% (w/v) + 4% sucrose + 200 mM HEPES solution for 15 min at 4°C.
• Wash with 1 ml PBS containing 0.1 M glycine five times.

Staining and Imaging

• Add Hoechst (λEx/λEm (with DNA) = 350/461 nm) at a final concentration of 1 µg/ml and WGA-Alexa Fluor 488 at a final concentration of 5 µg/ml (volume until coverslips are covered).
• Incubate for 5 min at RT in the dark.
• Remove and wash three times with PBS.
• Mount coverslips on glass slides with mounting reagent.
• Perform fluorescence or confocal fluorescence microscopy using appropriate excitation and emission filters.

Inter-Kingdom Conjugation Assay

Preparation of mammalian cells

• Seed HEK293T and HeLa cells in T25 flasks (7*10⁵ cells / flask), in 5 ml DMEM supplemented with 10% FCS, 2 mM L-glutamine and 1% penicillin/streptomycin and incubate in a humid CO₂ incubator at 37°C for 24 hours.

Preparation of bacterial strains

• Set up 5 ml overnight cultures with LB + appropriate antibiotics:
  • E.coli 10 beta with pHelper_RP4
  • E.coli 10 beta with pmob_m_CMV
  • E.coli 10 beta with pHelper_RP4 + pNeae2_7d12
  • E.coli beta with pmob_m_CMV + pNeae2_7d12
  • E.coli 10 beta with pHelper_RP4 + pmob_m_CMV (test group without adhesins)
  • E.coli 10 beta with pHelper_RP4 + pmob_m_CMV + pNeae2_7D12 (test group with adhesins)

Set up conjugation reaction

• 24 hours after seeding, aspirate media from T25 flasks containing HEK293T cells.
• Wash gently with 1x DPBS twice.
• Harvest bacteria by centrifuging at 13,000 rpm for 1 min.
• Resuspend the bacterial pellets in 5.5 ml DMEM supplemented with 10% FCS, 2 mM L-glutamine, 20 mM HEPES, 5 µg/ml DNaseI-XT and 1 µM cytochalasin D.
• Add 5 ml of the bacterial suspension (OD₆₀₀ 1.0) to the T25 flasks containing HEK293T cells - work outside the sterile hood from here to avoid contamination of the hood.
• Place in a bacteria incubator (static) at 37°C for 12 hours.

Elimination of bacteria after conjugation

• Remove media (containing bacteria) from the T25 flasks and wash 2-3 times with PBS supplemented with 150 mM NaCl + 50 µg/ml kanamycin.
• Add 5 ml of fresh DMEM supplemented with 10% FCS, 2 mM L-glutamine, 1% penicillin/streptomycin and 50 µg/ml kanamycin.
• Place in the bacterial incubator again for 2-3 h to neutralize remaining bacteria.
• Check under the microscope to ensure no prevailing signs of live bacteria.
• Thoroughly disinfect the exteriors of the flasks, then transfer to a humid CO₂ incubator at 37°C, incubate for 24 h.

Read-out

• Perform fluorescence microscopy for qualitative and FACS for quantitative readout of fluorescent reporter expression across the various experimental groups.