Experiments we performed

Plasmid Isolation

Media and Buffers:

• 2.5% LB BROTH - Mix 0.5 g of agar in 20 ml double distilled water. It was then autoclaved and stored at 4 degree celsius.
• 4% LB AGAR - 2g agar was added to 50ml double distilled water and then it was autoclaved and stored at 4 degree celsius for further use.

pET-28b+ transformed colonies were used for plasmid isolation:

• Two methods were used for the isolation of pET-28b+:
  1. Alkaline lysis method
  2. isolation using promega plasmid isolation kit

Inoculation of Bacteria

• Kanamycin plates with colonies of competent cells that were transformed with pET28b+ vector have been obtained from the iGEM 2021 team of IISER Berhampur.
• 5 ml of LB broth was taken in two, 15 ml Falcon tubes.
• 5 μl of kanamycin was added to each tube. (Since kanamycin stock concentration is generally 50mg/ml so to obtain a working concentration of 50μg/ml, we add 5μl of kanamycin in 5 ml broth).
• A single colony was inoculated in each tube.
• The tubes were incubated in a BOD incubator shaker under shaking conditions at 37 degrees Celsius at 162 rpm for 16 hours.

Here, Plasmid Isolation was done using these two process:

• Alkaline lysis method
• Using QIAGEN Kit

Alkaline lysis method

• After 16 hours of incubation, both the tubes containing LB broth were showing noticeable turbidity indicating bacterial growth.
• All centrifugation steps were carried out at 17000 rpm at 4 degrees Celsius.
• 5 ml bacterial overnight culture was pelleted by centrifugation at 17000 rpm for 1 min.
• Pellet was resuspended in ice cold 100microlitre alkaline solution Ⅰ by vigorous vortexing.
• 200 microliter of alkaline solution ⅠⅠ was added to it in RT and contents were mixed by inverting the tube several times and was stored in ice.
• 150 microlitre of ice cold alkaline solution ⅠⅠⅠ was added to this viscous bacterial lysate and was dispersed by inverting the tube.And store in ice for 3-5 minutes.
• Centrifuge the bacterial lysate for 5 minutes and collect the supernatant.
• Equal volume of phenol: chloroform (1:1) was added and the organic and aqueous phases were mixed by vortexing followed by centrifugation (2min). The aqueous upper layer was transferred to a fresh tube.
• Two volumes of ethanol was added to it and the solution was vortexed for mixing and let it stand in RT for 2 minutes for the nucleic acids to precipitate.
• Then it was centrifuged for 5 minutes at 17000 rpm and 4 degree Celsius and the precipitate was collected.
• The supernatant was discarded and the tube was kept in an inverted position to drain all fluids in the tube. In order to remove some drops on the walls of the tube pipette tip was used.
• 1 ml of 70% ethanol was added to the pellet and was inverted several times. The DNA was retained by centrifuging at maximum speed for 2 minutes at 4 degree Celsius.
• Supernatant was removed by aspiration (pellet won’t be tightly adhered to the tube in this step)
• Ethanol was removed from the tube and it was kept in the RT for the evaporation of ethanol until there was no fluid visible in the tube (Around 10 minutes).
• The pellet was then dissolved in 50 microlitres of TE buffer (pH 8.0).
• The tube was gently tapped for a few seconds and was kept at -20 degree Celsius.

Quantification of concentration and purity of the isolated plasmid

• Concentration and purity of the isolated plasmid DNA from colonies were quantified by a nanodrop spectrophotometer.
• TE Buffer was used for blanking.

Agarose gel electrophoresis protocol

Sample preparation

• Setup the gel tray
• Weigh 0.4 g of Agarose in a laboratory weighing balance.
• Dissolve 0.4 g of Agarose in 50 ml of 1X TAE in a 100 ml conical flask by heating in a microwave.
• Allow the dissolved agarose to cool down to a temperature that can be tolerated by touching on skin.
• Add 2.5 μl of Ethidium Bromide then swirled the conical flask to mix EtBr well and poured it on the gel tray.
• Allow the gel to harden (30 minutes)
• After the gel has hardened, the gel comb was withdrawn, taking care not to tear the sample wells.
• The gel casting platform containing the set gel was placed in the electrophoresis tank.
• Sufficient electrophoresis buffer (1X TAE)was added to cover the gel to a depth of about 1mm.
• Load 3 μl of 1Kb DNA Ladder in lane 1, 6 μl of C1 Sample in lane 3, and 6 μl of C2 Sample in Lane 5.
• The leads were attached so that the DNA will migrate into the gel toward the anode or positive lead.
• The voltage was set to 100V for 35 minutes.
• After the electrophoresis was completed the Gel was visualized under a UV Transilluminator.

Using QIAGEN Kit

• 2 ml of Bacterial overnight culture was pelleted by centrifugation at 13000 rpm for 3 min at room temperature (15–25°C) and the supernatant was discarded.
• The process was repeated till the whole 5ml of bacterial culture was pelleted.
• Pelleted bacterial cells were resuspended in 250 μl Buffer P1 and were transferred to a microcentrifuge tube
• 250 μl Buffer P2 was added and was mixed thoroughly by inverting the tube 4–6 times until the solution becomes clear. (Do not allow the lysis reaction to proceed for more than 5 min. If you use the LyseBlue reagent, the solution will turn blue.)
• 350 μl Buffer N3 was added and was mixed immediately and thoroughly by inverting the tube 4–6 times.(If using LyseBlue reagent, the solution will turn colorless.)
• The sample was centrifuged for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
• 800 μl of the supernatant was transferred to the QIAprep 2.0 spin column by pipetting.
• It was then centrifuged for 30–60 s (13000rpm) and discard the flow-through.
• The QIAprep 2.0 spin column was washed by adding 0.75 ml Buffer PE. Followed by Centrifugation for 30–60 s (13000rpm) and the flow-through was discarded.
• To elute DNA, we added 30 μl Buffer EB (10 mM TrisCl, pH 8.5), let it stand for 1 min, and centrifuged for 1 min.
• We added 1 volume of Loading Dye to 5 volumes of purified DNA and Mixed the solution well by pipetting before loading the Plasmid for analyzing on a gel.

Cloning and expression of fimH

• Gel Extraction
• PCR for primer resuspension
• Restriction digestion of Vector and Insert

Gel Extraction

• Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
• Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~100 µl).
• Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation.
• After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
• Add 1 gel volume of isopropanol to the sample and mix.
• Place a QIAquick spin column in a provided 2 ml collection tube.
• To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 minute.
• Discard flow-through and place QIAquick column back in the same collection tube.
• (Optional): Add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min.
• To wash, add 0.75 ml of Buffer PE to QIAquick column and centrifuge for 1 min.
• Discard the flow-through and centrifuge the QIAquick column for an additional 1 min at ≥10,000 x g (~13,000 rpm).
• Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
• To elute DNA, add 35 µl of Nuclease free water to the center of the QIAquick membrane and centrifuge the column for 1 min at maximum speed.

PCR for primer resuspension

In a sterile PCR tube, mix the contents in the following order:

Column 1	Column 2
Nuclease free Water	38μl
2x PCR master mix	50 μl
Template DNA (Plasmid)	4μl
FimH Forward primer	4μl
FimH Reverse primer	4μl
Total reaction volume	100 μl

• Mix the contents gently (pipette it), quickly centrifuge it and carry out amplification in the thermal cycler for 30 cycles using the following reaction conditions.



• Add 10 μl gel loading buffer.
• Carefully pipette out 50 μl reaction mixture and load in the well of the 0.8 % agarose gel.
• Loaded 3μl of the ready to use marker (1KB)
• Electrophoresis the samples at 100 volts for 1 to 2 hrs till the tracking dye (bromophenol blue) reaches 3/4 th of the length of the gel and the DNA marker is resolved properly
• Visualize the gel under UV transilluminator.

Restriction digestion of Vector and Insert

Prepare a reaction mix by adding reagents in the order indicated in the table below and Incubate at 37°C for 4 hours followed by a gel run.

Reagent	Volume
Nuclease free Water	10 μl
Tango buffer (10X)	5 μl
DNA (Vector)	2.5 μg
restriction enzyme 1 (Xho1)	1.5 µl
restriction enzyme 2 (Nco1 HFI)	1.5 μl
Total reaction volume	50 μl

Reagent	Volume
Nuclease free Water	8 μl
Tango buffer (10X)	5 μl
DNA (Insert)	35 μl
restriction enzyme 1 (Xho)	1.5 µl
restriction enzyme 2 (Nco HF)	1.5 μl
Total reaction volume	50 μl

Ligation of Vector and Insert

• Elute the vector backbone using a QIAquick Gel Extraction Kit Protocol following the above mentioned protocol. Elute the vector backbone in 30 ul of nuclease free water.
• Incubate at 37°C for 4 hours followed by a gel run. Purify the digested PCR product using QIA quick PCR purification kit protocol.
• Set up the following reaction in a microcentrifuge tube on ice. (T4 DNA Ligase should be added last.)

Reagent	Positive control	Negative Control
Vector	40 µg (~2.1 µl)	40 µg (~2.1 µl)
Insert	100 ng (3 µl)	---
T4 DNA ligase buffer 10X	2 µl	2 µl
T4 DNA ligase enzyme	1 µl	1 µl
Nuclease free water	11.9 µl	15 µl
Total volume	20 µl	20 µl

• Gently mix the reaction by pipetting up and down and microfuge briefly.
• For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.

Transformation

• Recover Dh5 alpha cells from -80°C.
• Put in ice for 5-10 minutes for thaw.
• Mix the contents of the tubes by swirling gently.
• Store the tubes on ice for 30 minutes.
• Transfer the tubes to a rack placed in a preheated 42°C water bath for heat shock. Keep the tubes in the rack for exactly 2 minutes. Do not shake the tubes
• Rapidly transfer the tubes to an ice bath. Allow the cells to chill for 5 minutes.
• Add 900 μl LB medium into the tube. Incubate the cultures for 45 minutes in a water bath set at 37°C to allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid.
• Centrifuge at 4000rpm for 3-4 minutes.
• Discard the supernatant but keep 100-200 ul for resuspension of pellet.
• Transfer the appropriate volume (up to 100 μl per 90-mm plate) of transformed competent cells onto agar LB medium containing kanamycin.
• Store the plates at room temperature until the liquid has been absorbed.
• Invert the plates and incubate at 37°C. Transformed colonies should appear in 12-16 hours.

Expression: Colony PCR

• Make LB agar + kanamycin plate
• Take 1.5 ml tubes.
• Add 50 µL 1X PBS in the tube. With the help of a tip take one colony and inoculate in tubes
• Then boil the sample at 95 degrees for 20 minutes. Spin it at 12,000 rpm for 10 minutes.
• Take 5µl plasmid as a positive control (plasmid - with the insert in this case).
• Similarly take 5 µL of H2O as a negative control.
• Prepare reaction mixture as following:
  • Template plasmid=5µL
  • Forward primer=1µL
  • Reverse primer=1µL
  • 2X PCR master mix = 12.5µL
  • H2O = 5.5µL
  • Total =25µL

Purification

Buffers required:

1. Lysis buffer- 50mM Tris, 200mM NaCl, 1mM PMSF, 1mMDTT, 10% Glycerol,, 10mM imidazole
2. Washing buffer- 50mM Tris, 200mM NaCl, 10% Glycerol, 20mM imidazole
3. Elution buffer - 50mM Tris, 200mM NaCl, 10% Glycerol, 200mM imidazole
• Store the pellet from -80 degree celsius and other components in ice.
• Prepare the lysate buffer by adding the components to a 50 ml falcon tube and make up the volume to 20 ml by adding milliQ water to it.
• Dissolve the pellet and pipette it several times and incubate for 15 minutes.
• Transfer it to a new falcon tube for sonication.
• Sonication : temperature-25 degree celsius; time- 1 hour; pulse 10:10 on:off
• Take the lysate and centrifuge at 12000 rpm for 10-15 minutes and collect the supernatant.
• Also take the beads and centrifuge at 13000rpm for 1 minute.
• Remove excess ethanol and mix the beads with the supernatant and incubate for 3 hours.
• Perform gel electrophoresis for quality check of the protein.
• Using a dialysis procedure, elute out protein.
• Concentrated protein using Amicon tubes and centrifugation process.

Future Experiment Planned

SELEX procedure

Buffers and solutions:

1. Binding buffer- pH 7.4 PBS containing 2.5 mM MgCl2 plus Salmon sperm ssDNA, 0.02% Tween 20 and 1 mM heparin. The amount of Salmon sperm ssDNA is 75 μg (equivalent to 3 nmol of 80 nt ssDNA) for round 1 and 5 μg (equivalent to 0.2 nmol of 80 nt ssDNA) for round 2 - 4, followed by 20 μg for rounds afterwards.
2. Wash buffer: pH 7.4 PBS containing 2.5 mM MgCl2, and 0.02% Tween 20.

Round One Selection

Initial ssDNA library preparation:

1. Add 30 μL of 100 μM ssDNA library (3 nmol) to 600 μL of binding buffer.
2. Mix and heat at 95°C for 10 min followed by chilling the denatured ssDNA on ice for 10 minutes.

Preparation of beads/protein complex:

3. For protein with molecular weight of 25 kDa, 4 μL (160μg based on 40mg/mL concentration) beads are enough to bind 0.1 nmol proteins for the first round SELEX.
4. Add 500 μL of binding buffer to beads in a 1.5 mL Eppendorf tube, place the tube on a magnet for 2 min, discard supernatant.
5. Prepare 500 μl of cell lysate containing overexpressed His-tagged protein target with equal volume of 2 ×Binding buffer and then mix well with beads.
6. Incubate mixture on a roller for 5 min at RT (or colder if the protein is unstable at RT).
7. Place the tube on the magnet for 2 min, discard the supernatant.
8. Repeat the wash 4 times each with a 300 μL wash buffer.
9. 10-20mM of imidazole can be added in the washing buffer to reduce the background nonspecific binding.
10. EDTA-free cell lysis buffers should be used since reducing agents such as Dithionite DTT and EDTA can enhance iron solubility and therefore pull the iron out of magnetic beads.
11. DNase I and 0.05% Tween 20 should be added to the lysate to help pelleting beads under the magnetic force and to minimize non-specific binding to plastic.



Test for checking binding capacity of beads to proteins by western blotting

1. The beads are resuspended in 1 × SDS PAGE loading buffer.
2. Then it is heated at 90 °C for 5 min and the supernatant is loaded on 10% SDS-PAGE gel.
3. The existence of interested protein on beads should be detected with specific primary antibodies following standard Western blot procedure.
4. The binding capacity can be calculated by Silver stain or Coomassie blue assay using protein standards.

Incubation of protein bait with the random DNA library:

1. The bead/protein complex is resuspended in 600 µL of binding buffer including 3 nmol of random ssDNA library and 1 mM heparin in a 1.5 mL Eppendorf tube.
2. The mixture is placed on a roller for 1 hour at RT to achieve good magnetic beads suspension.
3. After incubation, the tube will be placed on a magnet for 2 min, then discard the supernatant.
4. The beads are washed 5 times with 500 μL wash buffer, 5 min per wash.
5. The beads should be resuspended thoroughly between each washing step.
6. After the last wash, the bead/protein/ssDNA complex is resuspended in 100 μL TE buffer for preparative PCR assay.

Preparative PCR1: To determine the optimum cycle number of PCR required:

• PCR1 involves the standard PCR reaction at 50 µL/tube in 6 individual tubes (each for 8, 10, 12, 14, 16, 18 cycles) and one additional negative control (absence of DNA template).
• A 50 µL PCR amplification reaction volume.
• PCR thermal cycle parameters based on the melting temperature of the designed primers.

Steps:

• 4% agarose gel is prepared in 1 × TBE buffer.
• 5 µl sample for each cycle is loaded and electrophoresis is performed at 100 V for 40 min.
• The 8 PCR samples are imaged under a blue light trans-illuminator after GelStarTM stain.
• The cycle number that yields the strongest band at the expected position will be used for subsequent amplification PCR.

PCR2 to amplify the DNA pool for the next round of SELEX:

• After determining the optimized cycle number, amplification PCR will be conducted using the same PCR procedures as PCR1.
• To improve the SELEX efficiency, it is important to use all of the 100 µL beads/protein/ssDNA mixture for PCR2. Therefore, for the first round, a 2 µL DNA template per 50 µL reaction should be applied for PCR1.
• From round 2 onwards, a 1 µL ssDNA mixture should be used for PCR1 and a 500 - 1000 µL PCR2 reaction (need 10 – 20 µL elution) is generally enough to collect 0.3 nmol ssDNA for both subsequent round SELEX and binding assessment.

For subsequent rounds of selection, following the same steps as demonstrated for the second round, but with the following modifications as selection progresses.

• Gradually reduce the amount of protein baits from 0.1 nmol to 0.01 nmol.
• Gradually reduce the incubation time for ssDNA pool with target from 1 hour to 30 min.
• Increase the washing time and washing buffer volume.
• Gradually increase the amount of random sperm ssDNA from an equal amount of ssDNA pool to 10 times more.

Evaluation binding capacity

ELISA assay

• Add 1 µg of biotin conjugated anti-His antibody to 100 µL PBS and incubate with streptavidin-coated well at RT for 1 h.
• After washing with 200 µL PBS for 3 times, incubate the with 150 µL of either positive selection cell lysates or negative control cell lysates at 4 °C overnight.
• wash the wells with 200 µL PBS for 3 times and each well is blocked with 100µg of random herring sperm ssDNAs at RT for 1 h.
• After thorough wash with PBS, incubate the wells with 100 µl of 100 nM ssDNA pools at RT for 1h.
• wash wells with 200 µL/well wash buffer for 5 times.
• After incubation with HRP conjugated anti-FITC antibody for 1 h followed by thorough washing, the fluorescence intensity is determined with a plate reader after the addition of QuantaBlu Fluorogenic Peroxidase Substrate according to the manufacturer’s instruction.
• The selection completes when no further improvement in the binding capacity between pools from successive two or three rounds can be observed.

Validation: Whole SELEX

• Dissolve 1.0 µg of double-stranded DNA library, in 50 µL of PBS
• Then denature it by heating at 95 °C for 10 min and then cool on ice for 10 min.
• Incubate the denatured ssDNA with 100 µL of 1.0 × 10^8 cfu/mL of selected strain, with shaking (1500 rpm) at room temperature for 45 min
• Then remove the Unbound ssDNA by centrifugation at 8000 g for 10 min.
• After that elute the bound ssDNA in 500 µL of double distilled water (ddH2O) by heating bacteria–DNA complexes at 95 °C for 10 min.
• Then use the supernatants after the centrifugation for PCR amplification.
• Then do gel electrophoresis on 2.0% of agarose gel to confirm the purity of the PCR product.
• lso perform Counter selex rounds after 3 selection cycles.

Preparation of aptamer-functionalized AuNPs

• Add 9 μL 1 mM aptamers into a 2 mL centrifuge tube. Then 1μL acetate buffer (500mM，pH 5.2) and 1.5 μL 10 mM TCEP to it and incubate 1 h at room temperature.
• Add 3 mL AuNPs and shake slightly, incubate in a dark environment for 16 hours.
• Bring the solution into 300 μL Tris acetic acid buffer (500 mM，pH 8.2).
• Measure 500 μL solution and add into 2 mL centrifuge tube, followed by centrifugation for 15 min at 16110×g and remove supernatant.
• Resuspend the precipitate with Tris acetic acid buffer (25 mM，pH 8.2), and re-centrifuge at the same condition.
• Disperse the precipitate in each binding buffer.

References:

• Wang T, Yin W, AlShamaileh H, Zhang Y, Tran PH, Nguyen TN, Li Y, Chen K, Sun M, Hou Y, Zhang W, Zhao Q, Chen C, Zhang PZ, Duan W. A Detailed Protein-SELEX Protocol Allowing Visual Assessments of Individual Steps for a High Success Rate. Hum Gene Ther Methods. 2019 Feb;30(1):1-16. DOI: 10.1089/hgtb.2018.237