Introduction
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet.
Protocols
DNA Cloning Methods
Oligo annealing
The protocol is typically performed with oligos at a stock concentration of 100 µM and will yield 10 µM of annealed oligos, but can also be adjusted accordingly.
- Pre-heat a heating block to 95 °C
- Prepare the following oligo mixture in a 1.5 mL eppi
- 5 µL rCutsmart buffer
- 5 µL of each oligo to anneal
- Fill up to 50 µL with nuclease free water (35 µL)
- Incubate mixture for 5 min at 95 °C
- Turn of the heating block and leave eppi inside for at least 1 h
Alternatively the reaction can be done in a PCR cycler and the mixture cooled with 0.5 °C/s - Store at -20 °C until further use
Restriction Digest
Restriction endonucleases are commonly used to clone DNA fragments into a plasmid backbone. This protocol is based on High-Fidelity restriction endonucleases (New England Biolabs, Ipswich) but can be easily adapted for other endonucleases.
| Component | Volume/Amount |
|---|---|
| DNA | 1 µg |
| Restriction Endonucleases | 1 µL each |
| rCutSmart Buffer (10X) | 3 µL |
| Nuclease-free water | to 30 µL |
Incubate the reaction mixtures for 1 h at 37 °C.
Golden Gate Assembly
Golden Gate Assembly enables the efficient and precise assembly of multiple DNA fragments in a single reaction using type IIS restriction enzymes.
- Set up assembly reactions in PCR-tubes with 100 ng vector DNA as follows:
Component Volume/Ratio Vector:Insert 1:3 T4 DNA Ligase Buffer (10X) 2.5 µL T4 DNA Ligase 0.5 µL Type IIS Restriction Enzyme 1.5 µL Nuclease-free water to 25 µL - Place PCR-tubes in thermocycler and run the following program:
Step Temperature [°C] Time [min] Return to step # Passes total Activation of Restriction Enzyme 37 5 10 - 30 Activation of Ligase 16 5 1 10 - 30 Inactivation of Enzyme and Ligase 65 5 1 Hold 4 ∞ 1 - Store at 4 °C until further use.
Gibson Assembly
Gibson assembly is a molecular cloning strategy used to assemble multiple DNA fragments with overlapping overhangs. In contrast to classical restriction cloning, gibson assembly does not require restriction sites for assembly.
- Design DNA fragments with 15 - 20 bp overhangs (2 - 3 fragments) or 20 - 30 bp overhangs (4 - 6 fragments).
- Amplify DNA fragments via PCR.
- Set up Gibson assembly reaction mix:
Component 2 - 3 Fragment Assembly 4 - 6 Fragment Assembly DNA 0.02 - 0.5 pmol 0.2 - 1 pmol Gibson Assembly Master Mix (2X) 10 µL 10 µL Nuclease-free water to 20 µL to 20 µL - Incubate the reaction mix at 50 °C for 15 minutes (2 - 3 fragments) or 60 minutes (4 - 6 fragments).
Ligation
A ligation reaction joins a vector and an insert by linking their compatible ends using DNA ligase.
- Use 50 ng of vector DNA to set up ligation reactions in PCR-tubes as follows:
Component Volume/Ratio Vector:Insert Ratio 1:3 T4 DNA Ligase Buffer (10X) 2 µL T4 DNA Ligase 1 µL Nuclease-free water to 20 µL - Place PCR-tubes in the thermocycler and run at 16 °C for 20 minutes. Inactivate DNA ligase at 65 °C for 10 minutes.
KLD Ligation
KLD ligation is used to circularize blunt end DNA fragments. The KLD reaction mix contains a kinase, ligase, and DpnI restriction endonuclease.
- Set up the KLD reaction mixture (10 µL total volume):
Component Volume Blunt End DNA 1 µL KLD Reaction Buffer (2X) 5 µL KLD Enzyme Mix (10X) 1 µL Nuclease-free water 3 µL - Mix the reaction mixture by pipetting up and down.
- Incubate the reaction mixture at room temperature (25 °C) for at least 5 minutes.
- Use 5 µL of the reaction mixture for the transformation of 50 µL competent cells.
PCR
PCR (Polymerase Chain Reaction) is a technique used to amplify a template DNA by cycling through denaturation, annealing, and extension phases with a thermostable DNA polymerase.
- Set up assembly reactions in PCR-tubes for a total volume of 25 µL as follows:
Component Volume/Amount Template DNA 10 - 100 ng Forward Primer (10 µM) 1.25 µL Reverse Primer (10 µM) 1.25 µL Q5 High-Fidelity DNA Polymerase 0.5 µL Q5 Reaction Buffer (5X) 5 µL dNTP Solution Mix (10 mM) 1 µL Nuclease-free water to 25 µL - Mix gently and spin down.
- Place PCR-tubes in thermocycler and run the following program:
Step Temperature Time Return to step # Passes total Initial Denaturation 98°C 30 s 1 Denaturation 98°C 10 s 25 - 40 Annealing 60 - 72 °C 15 s 25 - 40 Extension 72°C 30 s/kb 2 25 - 40 Final Extension 72°C 5 min 1 Hold 4°C ∞ 1 - Store at 4 °C until further use.
Agarose Gel Electrophoresis
Agarose gel electrophoresis is used to separate DNA fragments of different nucleotide lengths from each other.
- Depending on the nucleotide lengths of the DNA fragments, gels with a different agarose concentration should be used:
Nucleotide Length Agarose Concentration 600 - 50000 bp < 1% 400 - 8000 bp 1% 100 - 2000 bp 2% 25 - 1000 bp 3% - Add agarose to TAE buffer in the desired agarose concentration and heat in the microwave until the agarose is completely dissolved (be careful to not let the agarose boil over).
- Add a staining agent to the agarose before pouring the cell (e.g. SYBR Safe).
- Pour gel and wait for 15 - 30 min until the gel becomes firm.
- Run gel at 10 volts/cm until the dye front has reached the bottom of the gel.
- Visualize gel under UV or blue light (depending on the staining agent used).
Competent Cells
Competent cells are bacteria that are able to take up DNA from their environment. Chemically competent cells are made competent with different salt solutions and can be transformed via heat shock.
Making Chemically Competent E. coli Cells
- To make chemically competent E. coli cells, inoculate 5 - 10 mL of LB medium with a single E. coli colony.
- Incubate the culture overnight at 37 °C while shaking until an optical density of 0.4 - 0.6 is reached.
- Transfer the culture in a sterile centrifuge tube and cool down on ice for 10 - 15 min.
- Centrifuge the culture at 4000 g for 10 min at 4 °C.
- Discard supernatant by decanting.
- Resuspend the cell pellet in an equal volume of cold, sterile 100 mM CaCl2 solution.
- Incubate for 30 min on ice, gently invert the tube every 5 - 10 min.
- Centrifuge cells at 4000 g for 10 min at 4 °C.
- Discard supernatant by decanting.
- Resuspend the cell pellet in 100 - 200 µL of cold, sterile 100 mM CaCl2 solution.
- Divide cells in aliquots (20 - 100 µL), flash-freeze in liquid nitrogen, and store at - 80 °C.
Transformation of Chemically Competent E. coli Cells
- Thaw cells on ice for 15 min.
- Add 1 - 2 µL of plasmid DNA to the cells and incubate for 30 min on ice. Do not mix.
- Heat-shock cells at 42 °C for 45 sec.
- Place on ice for 5 min.
- Add 1 mL of SOC outgrowth medium and recover cells for 45 min at 37 °C while shaking (Note: Recovery phase can be skipped if ampicillin is used as a selection marker).
- Centrifuge for 12000 rpm for 1 min.
- Discard supernatant by decanting and resuspend cell pellet in remaining supernatant..
- Plate bacteria on LB agar plates containing the appropriate antibiotics.
Protein Expression & Purification
Mammalian Cell Protocols
Cell Maintenance
This is a basic protocol for splitting Human Embryonic Kidney 293T (HEK293T) cells to maintain the culture throughout the week, with excess cells reserved for future experiments and plating. Make sure to work in a sterile environment to avoid contamination of the cells.
- Warm reagents to 37°C and prepare complete DMEM supplemented with 10% FCS and 1% P/S:
Reagent Volume [mL] Dulbecco's Modified Eagle Medium (DMEM) 445 Fetal Calf Serum (FCS) 50 Penicillin/Streptomycin (P/S) 5 - Take cells out of the incubator and aspirate the old media from the plate or flask.
- Wash with Dulbecco's Phosphate Buffered Saline (DPBS).
- Add trypsin and incubate at 37°C until cells start to detach.
- Add DMEM to neutralize the trypsin.
- Count cells or decide on the appropriate dilution based on confluence.
- Dilute with DMEM until the desired concentration is reached.
- Seed cells onto a new plate or flask, gently shuffle to ensure even dispersal, and return cells to the incubator.
Cell Seeding
This is a basic protocol for seeding Human Embryonic Kidney 293T (HEK293T) cells for future experiments, for example transfections. Make sure to work in a sterile environment to avoid contamination of the cells.
- Collect cells according to the instructions of the Cell Maintenance protocol.
- Count cells and calculate the appropriate dilution.
- Dilute with DMEM until the desired concentration is reached.
- Seed according to the cell culture dish you used:
Type of dish Seeding density Cells at confluency Volume of growth medium [mL] 6-well plate 0.3 x 106 1.2 x 106 2 - 3 12-well plate 0.1 x 106 0.5 x 106 1 - 2 96-well plate 0.01 x 106 0.04 x 106 0.1 - 0.2 T-75 flask 2.1 x 106 8.4 x 106 8 - 15 T-25 flask 0.7 x 106 2.8 x 106 3 - 5 - After seeding, gently shuffle to ensure even dispersal, and return cells to the incubator.
Transfection
Transfection refers to the uptake of exogenous DNA into eukaryotic cells.
- Seed cells in an appropriate well plate and incubate at 37 °C and 5% CO2 for 24 h before transfection (cells should reach a confluency of 70 - 90%).
- Dilute DNA to desired concentrations in OptiMEM medium (Thermo Fisher Scientific, Waltham).
- Dilute lipofectamine 2000 (Thermo Fisher Scientific, Waltham) transfection reagent in OptiMEM medium. Incubate for 5 min.
- Add lipofectamine to DNA solutions and incubate for 30 min.
- Add DNA-lipid complexes to cells and incubate cells at 37 °C and 5% CO2 for 1 - 3 days.
| Component | 96-well | 24-well | 6-well |
|---|---|---|---|
| Final DNA amount per well | 100 ng | 500 ng | 2500 ng |
| Final lipofectamine 2000 reagent per well | 0.2 - 0.5 µL | 1.0 - 2.5 µL | 5.0 - 12.5 µL |
Assays
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)
reparation 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
Table 2: Samples used to conduct the coIP experiment. Sample 1 - negative control (also to test cross-reaction of anti-myc beads to proteins in the HEK293T cell lysate); Samples 2 and 3 - verification of the presence of myc epitopes on the proteins of interest and anti-myc bead validation; Sample 4 - coIP (no nanobody control); Sample 5 - coIP (test group).
| 1 | 2 | 3 | 4 | 5 | |
|---|---|---|---|---|---|
| Lysate of HEK293T transfected with EGFR-GFP | + | - | - | + | + |
| Lysate of E. coli 10-beta with pNeae2 | - | + | - | + | - |
| Lysate of E. coli 10-beta with pNeae2_7D12 | - | - | + | - | + |
| anti-myc pulldown | + | + | + | + | + |
- 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.
Materials & Methods
Cas Staples
Heading 4
Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex! Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex! Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex! Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex! Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex! Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex! Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex!
Heading 4
Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex!
Heading 4
Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex!
Heading 4
Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex!
Heading 4
Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex!
Staple Functionalization
Molecular Cloning of the Cathepsin B-Cleavable Linker GFLG (p10)
We cloned the SV40-Gal4-GFLG-VP64 construct (p10) to evaluate whether the GFLG linker could be cleaved by cathepsin B in vivo. We purchased the GFLG linker as oligonucleotides (fwd_GFLG_p10_CatB, rev_GFLG_p10_CatB) for Golden Gate cloning. To prepare the backbone, we used a plasmid (p3661) already containing the SV40-Gal4-VP64 construct, which had a GS linker between Gal4 and VP64. Using primers fwd_Esp3I_CatB_p10 and rev_Esp3I_CatB_p10, we introduced Esp3I recognition sequences via overhangs and created the amplicon SV40-Gal4-VP64_backbone. The final plasmid assembly was carried out using Golden Gate cloning with the Type IIS restriction enzyme Esp3I, and the final plasmid was transformed into E. coli Top10 cells.
Molecular Cloning of the Cathepsin B Expression Cassette (p12)
We amplified the gene block gBlock_Cathepsin_B with the primers fwd_gBlock_CatB and rev_gBlock_CatB. The gene block was composed of an upstream XhoI recognition sequence, followed by a Kozak sequence, the SV40 nuclear localization signal, a GGS linker, the human codon-optimized sequence for cathepsin B, and a downstream BamHI recognition sequence. We then inserted the amplicon into a pcDNA3.1 backbone via restriction cloning and transformed the ligated plasmid into E. coli Top10 cells.
Molecular Cloning of a Truncated and Mutated Form of Cathepsin B (p12.1)
To generate a truncated and mutated version of cathepsin B, we employed overlap extension PCR. We used the primers CatB_p12_frag1_fwd and CatB_p12_frag1_rev to remove the nucleotide sequence corresponding to the first twenty amino acids of wild-type cathepsin B using the gene block gBlock_Cathepsin_B as a template. We introduced three point mutations (D22A, H110A, and R116A) in the nucleotide sequence of cathepsin B using the primers CatB_p12_frag2_fwd, CatB_p12_frag2_rev, CatB_p12_frag3_fwd, CatB_p12_frag3_rev, CatB_p12_frag4_fwd, and CatB_p12_frag4_rev. After generating the amplicon (trunc_mut_CatB) with overlap extension PCR, we cloned it into the same pcDNA3.1 backbone used for the wild-type cathepsin B and transformed the ligated plasmid into E. coli Top10 cells.
Cathepsin B Cleavage Fluorescence Readout Assay
To study cathepsin B-mediated cleavage of different linkers, we conducted a fluorescence readout assay. We seeded 104 HEK293T cells in each well of a 96-well plate with DMEM (10% FCS) and incubated the cells for 24 hours at 37 °C and 5% CO2 before transfection. Plasmid solutions were prepared in OptiMEM, and transfections were performed using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham). For each linker, we included one negative control without the plasmid encoding cathepsin B, and two test samples with 30 ng and 60 ng of the plasmid. After transfection, we incubated the cells at 37 °C and 5% CO2. At 24 hours post-transfection, we added 500 nM doxorubicin to the cell supernatant. We measured eGFP and mCherry fluorescence intensities at 48 hours post-transfection using the Tecan Infinite F200 Pro plate reader (Tecan Group Ltd., Männedorf).
Cell Lysis and Western Blotting
To confirm the overexpression of cathepsin B in HEK293T cells, we performed western blotting. We seeded 105 HEK293T cells per well in a 12-well plate with DMEM (10% FCS) and incubated for 24 hours at 37 °C and 5% CO2 before transfecting cells separately with both wild-type and mutated cathepsin B constructs. Transfections were carried out in OptiMEM using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham), followed by incubation at 37 °C and 5% CO2. At 24 hours post-transfection, we supplemented the medium with 500 nM doxorubicin. 48 hours post-transfection, we washed the cells with DPBS. We then lysed the cells in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% Sodium deoxycholate, 0.1% SDS, 1 mM dithiothreitol, 50 mM TRIS-HCl pH 7.6) supplemented with a protease inhibitor cocktail. Protein concentrations were quantified using the DC protein assay (Bio-Rad Laboratories GmbH, Feldkirchen) and measured with the Spark Multimode microplate reader (Tecan Group Ltd., Männedorf). For SDS PAGE, we used a Mini-PROTEAN TGX Stain-Free Precast gel (Bio-Rad Laboratories GmbH, Feldkirchen) and ran the gel in a Mini-PROTEAN Tetra cell system (Bio-Rad Laboratories GmbH, Feldkirchen) at 200 V for 30 minutes. Following SDS PAGE, we transferred proteins to a PVDF membrane via semi-dry western blotting using the Trans-Blot SD system (Bio-Rad Laboratories GmbH, Feldkirchen) at 10 V for 1 hour. The membrane was blocked with I-Block protein-based blocking reagent (Thermo Fisher Scientific, Waltham) and incubated overnight at 4 °C with a polyclonal anti-cathepsin B antibody and a monoclonal anti-beta-tubulin antibody (Proteintech, Rosemont) (1:1000 dilution). After washing thrice with TBS-T buffer, we incubated the membrane with anti-rabbit IgG DyLight 800 (Cell Signaling Technology, Inc., Danvers) and anti-mouse IgG Alexa Fluor 680 (Thermo Fisher Scientific, Waltham) (1:10000 dilution) for 1 hour at 4 °C. Afterwards, we washed the membrane three times with TBS-T buffer and imaged the membrane using the Odyssey XF Imager (LI-COR Biotechnology, Lincoln).
Molecular Cloning of an Intein-dCas9 Fusion Protein (p13)
To enable functionalization of Cas staples in vivo, we cloned a dCas9-intein fusion protein using HiFi DNA assembly. We PCR-amplified the inteins NpuC and NpuN from the gene block gBlock_dCas9_Inteins using the primers SV40_NpuC_Cage_fwd, SV40_NpuC_rev, NpuN_SV40_fwd, and NpuN_Cage_SV40_rev. We amplified the backbone (p70) and dCas9 (p505) with the primers p70_BB_SV40_fwd, p70_BB_SV40_rev and dCas9_fwd, dCas9_rev creating the amplicons p70_backbone and dCas9, respectively. Finally, we assembled the fragments using the NEBuilder HiFi DNA assembly reaction mix (New England Biolabs, Ipswich).
Readout System
Heading 4
Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex! Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex! Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex! Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex! Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex! Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex! Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex!
Heading 4
Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex!
Heading 4
Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex!
Heading 4
Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex!
Heading 4
Lorem ipsum dolor sit amet consectetur adipisicing elit. Quas, veniam. In a voluptatum consequatur. Tempora, id! Hic, vero, similique dolore porro distinctio quasi minus consequuntur nobis officia voluptatum qui ex!
Delivery System
Cloning of pHelper_RP4 (Helper Plasmid Encoding the RP4 Conjugation Machinery)
pHelper_RP4 was constructed from pTA-Mob 2.0 (addgene #149662) by removing the oriT sequence and inserting the gene block (DSg1) using restriction-ligation cloning. The oriT was removed by digesting the plasmid with SpeI and SacI, and the linearized plasmid was gel-purified using the Large Fragment DNA recovery kit (Zymo Research). Meanwhile, a gene block encoding the traJ region that was removed and containing the SpeI and SacI restriction sites (DSg1) was PCR amplified, digested with SpeI and SacI and subsequently cleaned up. Then, overnight ligation (at 16°C) of linearized pTA-Mob 2.0 and restricted DSg1 with T4 DNA ligase was set up.
Cloning of pmob_b (Mobilizable Plasmid for Conjugation Between Bacteria)
The pSC101 ori of 2374_pSC101 was swapped with p15A ori from pRep_p15A to generate pmob_b. The backbone for pmob_b was amplified via PCR from 2374_pSC101 with the primers DS3 and DS4. The p15A origin of replication (ori) was amplified via PCR from pRep_p15A with the primers DS5 and DS6. In both cases, the primers were designed to produce the necessary homology regions for subsequent utilization in Gibson Assembly.
Cloning of pmob_m_CMV (Mobilizable Plasmid for Conjugation between Bacteria and Mammalian Cells)
pmob_m_CMV was cloned using four-fragment Golden Gate Assembly. The two parts of the backbone were amplified via PCR from pcDNA3.1 respectively with the primer pairs DS7, DS8 and DS11, DS12. The p15A ori was amplified via PCR from pRep_p15A with the primers DS9 and DS10. The origin of transfer (oriT) sequence was amplified via PCR from 2374_pSC101 with the primers DS13 and DS14. All resultant PCR amplicons possessed 5’ and 3’ extensions with BsmBI recognition sites and were designed to be assembled directionally after cleavage by BsmBI. Following gel extraction, the four fragments were then assembled via Golden Gate Assembly using BsmBI.
Cloning of pmob_m_CEA (Mobilizable Plasmid for Conjugation Between Bacteria and Mammalian Cells)
pmob_m_CEA was cloned from pmob_m_CMV by exchanging the CMV-promoter with the CEA-promoter. The backbone was amplified from pmob_m_CMV via PCR with the primers DS15 and DS16. The CEA-promoter was ordered as a gene block (DSg2) and amplified via PCR with the primers DS17 and DS18. Both resultant PCR amplicons carried 5’ and 3’ extensions with BsmBI recognition sites and were designed to be assembled directionally after cleavage by BsmBI. PCR product clean-up was performed for the amplified CEA promoter and the PCR product of the pmob_m_CMV backbone was gel purified. The promoter and the backbone were then assembled via Golden Gate Assembly using BsmBI.
Cloning of pNeae2_7D12 (for Surface Expression of Synthetic Adhesin Against EGFR on E. coli 10-beta)
The cloning of pNeae2_7D12 will be done by two-fragment Gibson Assembly using pNeae2 (addgene #168300) as the backbone. The backbone will be amplified using the primers - DS19 and DS20. The gene block encoding wt 7D12 adhesin (DSg3) will be amplified using the primers - DS21 and DS22. The PCR amplicons are designed to possess the necessary homology regions for Gibson Assembly. Following clean-up, the PCR amplicons will then be assembled via Gibson Assembly.
Solid Media Bacterial Conjugation Assay
In preparation for the conjugation assay, the corresponding plasmids were transformed into respective chemically competent E. coli strains and plated on appropriate antibiotic-containing LB agar plates to create the necessary donor and recipient cells. Single colonies were picked and 5 ml overnight cultures were set up in LB medium supplemented with the appropriate antibiotics.
The experimental design involved a positive control, two negative controls, and a test group, as outlined in Table 1 below. In all cases, the recipients were made to carry a plasmid conferring resistance against ampicillin (pAmpR) in order to enable selection against donors.
Table 1: Donors and recipients in the different controls and in the test group used for the bacteria-bacteria conjugation assay.
| Donor | Recipient | |
|---|---|---|
| Negative control 1 | E. coli 10-beta with pHelper_RP4 | E. coli BL21(DE3) |
| Negative control 2 | E. coli 10-beta with pmob_b | E. coli BL21(DE3) |
| Positive control | - | E. coli BL21(DE3) transformed with pmob_b |
| Test group | E. coli 10-beta with both pHelper_RP4 and pmob_b | E. coli BL21(DE3) |
The following day, 500 µl of liquid cultures were reserved as glycerol stocks and stored at -80 °C for later use. The remaining liquid bacterial cultures were then centrifuged in separate 1.5 ml tubes at 11,000 rpm for 1 min and the pellets were resuspended in 100 µl of 10 mM magnesium sulfate solution (diluent) as suggested in literature (Silbert et al., 2021). The optical density (OD600) was adjusted to 10 using the diluent. The experimental groups were then prepared for the conjugation assay by mixing together 100 µl of the respective OD-adjusted recipient and donor cell suspensions. To each tube, 1 ml of diluent was then added, the tubes vortexed and centrifuged for 1 min at 11,000 rpm. After removal of the supernatant, the cell pellets were resuspended in 10 µl of the diluent and mixed gently. The 10 µl drop was then pipetted onto an antibiotic-free LB agar plate and let air dry for 10 min. Afterwards, the plates with 10 µl bacterial spots were incubated at 37 °C for 18 hours, after which the bacterial patches were collected using sterile inoculation loops, resuspended in 1 ml of diluent and the OD600 of all suspensions were adjusted to 2.4. Serial dilutions were then prepared in the diluent using the OD-adjusted cell suspensions and the dilutions ranged from 10-2 to 10-9. The dilutions were plated on two types of LB agar plates - one with only ampicillin (selects for recipients) and another with both ampicillin and kanamycin (selects for transconjugants). Three technical replicates were used for the test group. Plates were then incubated overnight at 37 °C.
Liquid Media Bacterial Conjugation Assay
Bacteria from the same clones used for conjugation on solid media were used for conjugation on liquid media. The same steps as for solid media conjugation were followed until the first resuspension step. The first resuspension was performed in 1 ml of antibiotic-free LB medium, followed by OD600 adjustment (to 10). For each experiment group, 120 µl of the corresponding OD-adjusted liquid cultures were mixed together and then centrifuged for 1 min at 11,000 rpm. After resuspension of the pellets in 1.2 ml of LB medium each, 1 ml of cell suspension from each experiment group was pipetted into a well of a 12-well plate and incubated at 37 °C for 18 hours without shaking. After incubation, the bacterial suspensions were removed from the wells and their OD600 was adjusted to 2.4 using the diluent. Serial dilutions were prepared and plated on selective agar plates (same as for solid media conjugation). Three technical replicates were used for the test group. The plates were then incubated overnight at 37 °C.
In both solid and liquid media conjugation assays, following the last overnight incubation step, the conjugation efficiency was calculated by counting colonies at a certain dilution on the ampicillin+kanamycin plate (selecting for transconjugants) and dividing them by the number of colonies on the corresponding ampicillin plate (selecting for total number of recipients).
Sequences
Lorem ipsum dolor sit amet, consetetur sadipscing elitr, sed diam nonumy eirmod tempor invidunt ut labore et dolore magna aliquyam erat, sed diam voluptua. At vero eos et accusam et justo duo dolores et ea rebum. Stet clita kasd gubergren, no sea takimata sanctus est Lorem ipsum dolor sit amet. Lorem ipsum dolor sit amet, consetetur sadipscing elitr.
Lorem ipsum dolor sit amet consectetur adipisicing elit. Corrupti doloremque necessitatibus, praesentium fuga labore ipsum dolores architecto laudantium voluptatum dolorem eius ducimus porro velit expedita non sint illo sequi rem.