Protocols

These settings are used for analysing the fluorescence of fuGFP, sfGFP and eGFPs. fuGFP excites 400 nm, while sf and eGFP excites at 470 nm. See the appropriate parts pages for more information. 20 flashes per well.

1. Calculate equimolar volumes of GFPs to Nanobody-surface display constructs. Purified stocks of GFPs at 1 mg/mL, equivalent to roughly 2.0✕10¹⁶ molecules per mL (see materials for more details). Surface display construct is around 6000-8000 molecules per bacterium (Salema & Fernandez 2017). Assuming OD1 is equivalent to around 2.5 ✕ 10⁹ cells per mL, there is roughly 2.0 ✕ 10¹³ molecules per mL. Therefore at OD1, it is a 1:1000 dilution for equimolarity.
2. Measure ODs of cell broths. Adjust calculations as above to determine dilution of GFPs and/or volume of cells to maintain approximate equimolarity. Aim for between 400 μL and 750 μL of diluted GFP solution to be added to each tube.
3. Divide cells into 1 mL aliquots in Eppendorf tubes. Pellet cells (2 minutes at max). Discard supernatant.
4. Wash in PBS: Resuspend in 500 μL of PBS, thoroughly homogenize (vortex or pipette). Pellet cells (30 seconds at max).
5. Resuspend in GFPs diluted in PBS buffer (according to above equimolarity calculations). Aim for a volume between 400 μL and 1 mL.
6. Incubate on a shaker for 15 minutes at 37 °C.
7. Pellet cells (1 minute at max). Discard supernatant - use a pipette. Pouring off supernatant results in drops stuck to the inside of the tube (it is important that as close to all GFP not bound to nanobodies are washed away).
8. Add 500 μL of PBS. Resuspend thoroughly (vortex or pipette).
9. Pellet cells (30 s at max). Discard supernatant using a 200 μL (or equivalent) pipette. 1 mL pipette tips are too wide to remove all supernatant without disturbing the pellet.
10. Repeat steps 8 and 9 two more times for three washes in total.
11. Resuspend in 500 μL - 1 mL of PBS (depending on size of pellet). Make sure cells are well-homogenized and no clumps remain.
12. Load 50 μL into the plate reader to measure fluorescence. Blank with PBS.

Reference

Salema, V. and Fernández, L.Á., 2017. Escherichia coli surface display for the selection of nanobodies. Microbial biotechnology, 10(6), pp.1468-1484.

This protocol resumes after cloning and transforming the shuffled PCR products into the desired vector and organism.

Recovery of Library

1. Pipette 1-2 mL of LB onto plated transformations (depending on number of colonies).
2. Scrape with the flat side of the spreader to collect all colonies.
3. Tip the plate (about 20 °C) and use a pipette to draw out liquid. Deposit into a large falcon tube. Repeat until all liquid is recovered.
4. Repeat for all plates of transformations.
5. Mix cell suspension thoroughly (vortex) to remove all clumps. A homogenized cell suspension is critical at this stage. Each 1mL is ideally a representation of all diversity present in the library.
6. Glycerol stocks of 1 mL aliquots of the library can be made and frozen. (E.g. Pellet 1 mL of cells and resuspend in 500 μL of PBS, then add 500 μL of 50% glycerol). Functionally these stocks should be identical in their representation of diversity of the library.

Screening of Library

1. Inoculate 50 mL LB broth with 1 mL of recovered cells. Induce with 100 μM cumate overnight.
2. Prepare the cotton plug. Soak approximately 100-130 mg of cotton in either GFP-CBD lysate diluted, pelleted and resuspended in PBS and filter sterilised. Note that all materials must stay sterile. Use of a biosafety cabinet or working in front of a bunsen is recommended.
3. Incubate cotton in GFP lysate on a shaker for 10 minutes.
4. Prepare the column. Remove the plunger of a 10 mL syringe. Transfer the cotton plug into the syringe, and plunge to form a dense plug.
5. Wash off unbound GFP-CBDs by adding 10 mL of PBS to the column. This can be repeated until the flow-through is clear but the cotton plug remains fluorescent green under UV. Flow-through can be collected for OD analysis.
6. Add cells to the column 10 mL at a time. Most cells should flow through the column - if not, the plug density may need to be adjusted.
7. Wash extremely stringently with PBS. Adding 10 mL at a time, collect flow through for OD analysis every few or so washes. Continue adding PBS until the OD off the flow through is zero.

Enrichment of Screened Nanobodies

1. Flame tweezers, retrieve cotton from the column and place into a 50 mL falcon with 10-25 mL of LB.
2. Incubate on a shaker at 37 °C. Grow for two growth cycles (around an hour).
3. Plate at an appropriate dilution (based on OD) to enable growth of individual colonies on the plate.

This is protocol is basd on the manufacturer’s protocol for 2x MangoMix provided by Bioline.

1. On the plate you are taking samples from, identify around 20 well isolated colonies and number them with a marker.
2. On an agar plate of the appropriate media for your organism, draw a grid and number the squares so you have one square for each sample. The squares should be 1-2 cm in size.
3. Combine the following on ice in a 1.5 mL Eppendorf tube to create a master mix (the following volumes are for 1x, multiply the volumes by the number of reactions you are doing):
   • 9.6 µL MilliQ H₂O
   • 10 µL 2x MangoMix
   • 0.2 µL 50 µM forward primer
   • 0.2 µL 50 µM reverse primer
4. Still making sure to keep everything on ice, aliquot 20 µL of the master mix into PCR tubes.
5. Using a 10 µL pipette tip, touch one of your numbered colonies. When doing this, make sure you get a very small amount of colony, it should be such a small amount that it is barely visible. Then take this tip and dip it a few times into the corresponding PCR tube. After this, use the tip to scratch 3 parallel lines in the corresponding square in your labeled agar plate.

This protocol is a slightly altered version of a previously published protocol (Meyer et al. 2015). We made some minor changes to fit our project but the basic process is unchanged from the original paper.

Before beginning DNA shuffling, you need to design two sets of primers. One is the outer set of primers, which should bind well outside (~50 bp on either end) of your region of interest. These will be used in the initial amplification of your fragments. During the process of DNA shuffling, the ends of the fragments will degrade, and the final PCR will need to use primers that amplify closer to the area of interest. These primers will be the inner set of primers.

Preparation of Linear Input DNA

1. Generate double-stranded DNA versions of the target regions using PCR amplification with the outer set of primers.
2. Purify PCR product using spin-column purification (see Coleman Lab basic protocol). 2 µg of total DNA is required for the rest of the shuffling protocol.

Fragmentation and Purification of Fragments

3. Create a 10x DNAse buffer by combining equal parts of 1 M Tris-HCl pH 7.4 and 200 mM MnCl₂. This must be done the day of the experiment because the final solution quickly degrades.
4. Combine the following in a PCR tube:
   • 5 μL 10X DNase I buffer
   • 2 μg Linear Input DNA (equal parts of each variant)
   • Water to bring total volume to 50 μL
5. Equilibrate the above reaction at 15 °C for 5 minutes in a thermocycler.
6. Add 0.5 μL DNase I (diluted to 1 U/μL in 1X DNase I buffer) to the mixture.
7. Incubate the reaction at 15 °C for 3 minutes.
8. Stop the reaction by incubating at 80 °C for 10 minutes.
9. Perform a PCR clean-up using spin column purification (see Coleman Lab basic protocol).

Reassembly

10. Combine the following in a PCR tube:
    • 200 ng DNA fragments
    • 2 units of a mixture of equal parts Q5 polymerase and taq polymerase
    • 10 μL 600 mM Tris-SO4 (pH 8.9), 180 mM Ammonium Sulfate
    • 5 μL 4 mM dNTPs
    • 4 μL 50 mM MgSO₂
    • Water to 100 μL
11. Thermocycle above reaction as follows: 94 °C for 2 minutes; 35 cycles of (94 °C for 30 seconds, 65 °C for 90 seconds, 62 °C for 90 seconds, 59 °C for 90 seconds, 56 °C for 90 seconds, 53 °C for 90 seconds, 50 °C for 90 seconds, 47 °C for 90 seconds, 44 °C for 90 seconds, 41 °C for 90 seconds, 68 °C for 90 seconds per kb) 68 °C for 2 minutes per kb.
12. Perform a PCR cleanup via spin column purification (see Coleman Lab basic protocols).

Reamplification

13. PCR amplify 5 μL (one-tenth) of the elution reassembly product using a thermostable, proofreading DNA polymerase and the inner set of primers.
14. Perform a PCR cleanup via spin column purification (see Coleman Lab basic protocols).

Reference

Meyer, A. J., Ellefson, J. W., & Ellington, A. D. 2014. Library generation by gene shuffling. Current protocols in molecular biology, 105, Unit–15.12. https://doi.org/10.1002/0471142727.mb1512s105

1. Inoculate 5 mL LB broths with a single colony from an agar plate.
2. Add kanamycin (50 µg/mL) and cumate (100 µM).
3. Incubate at 37 °C, 200 RPM overnight (~18 h).
4. Load cells in LB 1 mL at a time into 2 mL beat beating tubes filled with 2 large glass beads, 3 scoops of medium-sized glass beads, and 2 scoops of small glass beads and centrifuge at 13,400 RPM for 30 seconds to pellet.
5. Add 400 µL TE buffer into the bead beating tube.
6. Bead beat at 3,000 RPM for 3 minutes.
7. Centrifuge bead beating tubes at 13,400 RPM for 5 minutes and collect the supernatant cell lysate in a new Eppendorf tube.

1. From a single colony from an agar plate inoculate 5 mL LB with kanamycin (50 µg/mL) and incubate overnight (~18 h) at 37 °C, 200 RPM to make a starter culture.
2. Add kanamycin (50 µg/mL) to 100 mL LB.
3. Inoculate 100 mL LB with 1 mL of starter culture.
4. Incubate at 37 °C, 200 RPM until OD₆₀₀ reaches ~0.6-0.7 (around 2-3 hours).
5. Induce culture with IPTG (0.1 mM).
6. Incubate at 37 °C, 200 RPM for 6 hours.
7. Remove culture, centrifuge 3,900 RPM for 10 mins to pellet cells and discard LB.
8. Resuspend the total 100 mL worth of cell pellet in a total of 3 mL of NT buffer. (Residual volume gives a total of ~4 mL cell resuspension).
9. Load cell resuspension 1 mL at a time into four 2 mL beat beating tubes filled with 3 large glass beads, 3 scoops of medium-sized glass beads, and 2 scoops of small glass beads and centrifuge at 13,400 RPM for 30 seconds to pellet.
10. Add 500 µL NT buffer into each bead beating tube.
11. Bead beat at 3,000 RPM for 3 minutes.
12. Centrifuge bead beating tubes at 13,400 RPM for 5 minutes and collect the supernatant cell lysate in a new test tube.
13. *Optional: check remaining cell pellet in bead beating tube under UV, if significantly green then repeat steps 10-12 with 250 µL NT.

1. Add 100 µL of cell lysate containing fuGFP-CBD to a spin column with a small disk of filter paper.
2. Vortex the spin column and incubate for 1 hour at room temperature.
3. Centrifuge the spin column at 13,900 RPM for 30 s and collect the flow through in a separate Eppendorf tube.
4. Add 200 µL of NT buffer to the spin column.
5. Centrifuge the spin column at 13,900 RPM for 30 s and collect the wash fraction in a separate Eppendorf tube.
6. Repeat steps 4-5 twice more.
7. Remove the filter disk from the spin column for analysis.
8. Analyse samples for fluorescence (excitation 400 nm, emission 520 nm) or using SDS-PAGE.

1. Set up cellulose ‘columns’ in a 1.5 mL Eppendorf tube by adding 250 uL of microcrystalline cellulose well suspended in NT (0.1 g/mL).
2. Centrifuge cellulose tubes at 13,400 RPM for 1 min and remove supernatant.
3. Load the cellulose column by adding 250 µL of cell lysate containing fuGFP-CBD fusion proteins.
4. Vortex well and incubate for 1 h at room temperature with rotation.
5. Centrifuge the column at 13,400 RPM for 1 min and remove supernatant (sample).
6. Wash the tube by adding 250 µL NT.
7. Vortex well then centrifuge at 13,400 RPM for 1 min and remove wash supernatant (sample).
8. Repeat steps 6-7 twice more.
9. Add 250 µL of 1 M glucose to the cellulose column tube.
10. Vortex well, centrifuge at 13,900 RPM, and collect supernatant (elution fraction).
11. Repeat steps 9-10 at least 3 more times or until the elution fraction is not fluorescent under UV.
12. Analyse samples for fluorescence (excitation 400 nm, emission 520 nm) or using SDS-PAGE.

These settings are used for analysing the fluorescence of fuBFP. fuBFP excites 380 nm. See the appropriate Parts page for more information. 51 flashes per well.

These settings are used to measure the excitation and emission spectra of fuGFP and its variants.

20 flashes per well, top optic.

Excitation scan: excites from 341-520 nm and measures emission at 554 nm. Gain 991.

Emission scan: excites at 445 nm and measures emission from 481-560 nm. Gain 693.

These settings are used to measure the excitation and emission spectra of fuBFP.

20 flashes per well, top optic.

Excitation scan: excites from 320-415 nm and measures emission at 460 nm. Gain 1200.

Emission scan: excites at 370 nm and measures emission from 401-514 nm. Gain 1200.