Experiments

Cloning Strategies

The final objective was to clone a small library of metal-binding proteins to evaluate their capacity to bind metals. Two distinct cloning strategies were employed, contingent on the length of the protein. The sequence was introduced via a PCR devoid of restriction enzymes for peptides comprising fewer than 50 amino acids (aa). In contrast, for proteins exceeding 50 aa, the restriction enzyme cleavage site was initially introduced via a PCR, followed by a Golden Gate assembly. Note that all our enzymes came from New England Bioscience. The complete list of proteins is available on the Parts page .

Plasmid Extraction

DH10B cells harboring the pBAD_sfGFP were grown overnight with kanamycin (50 µg/mL) in a Luria-Bertani (LB) medium. It contains an inducible araBAD promoter followed by sfGFP carrying Kan resistance gene. The tubes were centrifuged at 5000 x g for 15 minutes at 4°C. The pellet was recovered. Following the manufacturer's procedure, the plasmid was extracted from the cells using a Thermofisher Scientific GeneJET Plasmid Miniprep Kit . The dsDNA concentration and the purity rations (260/230 and 260/280) were measured using the SPECTROstar Nano (BMG Labtech). Prof. Vitor Pinheiro donated the empty backbone plasmid.

Primers Design

The primers were designed using Benchling and ordered from Integrated DNA Technologies (IDT). Recognition sites for BsaI-HF®v2 or PaqCI® were added. Overhangs were incorporated depending on the cloning strategies used. For instance, to perform a Gibson assembly, the overhangs of the primer used to amplify our insert are homologous to the overhang of the primer used to amplify our pBAD. The annealing temperature Tm was calculated using the NEB calculator.

A complete spreadsheet of all primers used can be found in the pdf here

PCRs

We amplified our short peptides, gBlocks™, linker and plasmids using Taq DNA Polymerase (NEB) and Q5 High Fidelity DNA Polymerase (NEB). These PCR reactions were conducted according to the manufacturer’s guidelines:

Table. 1 PCR Components and Volumes for 25 µl and 50 µl Reactions Using Taq DNA Polymerase with Standard Buffer

PCR Components and Volumes for 25 µl and 50 µl Reactions Using Q5 High-Fidelity DNA Polymerase

Gel Electrophoresis

Gel electrophoresis was carried out to separate and visualize our amplified DNA based on its size. It is also a way to assess the success of our PCR. Depending on the length of our DNA fragments, we prepared agarose gel with the optimal resolution according to the manufacturer’s labels. The procedure is described as follows:

1. Mixing the adequate amount of agarose powder with 50ml of 1X lithium acetate.
2. The mixture must be microwaved until complete dissolution.
3. Add 3 μL SBYR safe and pour everything into a gel tray with the well comb in place. Make sure there are no bubbles and wait until gel solidification at room temperature.

The next step is sample loading which is performed as follows:

1. 5 uL of DNA was added to 1 uL of 6X Loading buffer. The sample was then collected and loaded in the wells.
2. For our shortest peptides, we used a Low Molecular Weight DNA Ladder. Whereas, for our gBlocks™ and plasmids, we used either a 1 kb DNA Ladder or a 1 kb plus DNA Ladder. All ladders were acquired from NEB.

DpnI digestion

Once our PCR products were ready, the methylated strands were digested with DpnI for 1 hour at 37°C to select for the newly synthesized DNA strands.

DNA Purification

We extracted our PCR products from the reaction using a GeneJET PCR purification kit from Thermo Fisher Scientific according to the manufacturer's instructions. The principle behind this procedure is that the mixture containing DNA is mixed with the binding buffer and passed through the GeneJET Purification Column. The binding buffer contains a chaotropic agent that denatures proteins and promotes the binding of the DNA to the silica membrane inside the column. Any impurities are removed in the washing step. The purified DNA is eluted from the column using the elution buffer. Finally, the quantity and the quality of the purified DNA is assessed using the SPECTROstar Nano LVis Plate (BMG LABTECH).

Phosphorylation

44 uL of the digested purified DNA is mixed with 1 uL of T4 PNK and 5 uL of 10X Buffer DNA ligase (10 mM ATP). The mixture is then incubated at 37°C for 1hr and heat inactivated by incubating at 80°C for 20 minutes.

Ligation

In our 55 uL sample, 1 uL of the T4 DNA ligase is added. The sample is incubated for 2 hours at room temperature or kept in the thermocycler overnight at 16°C.

Transformation

After obtaining our correct genetic material, chemically competent DH10B cells were used (see below). 50 uL of competent cells that were previously stored at - 80°C were thawed on ice and 5 uL of the cloning reaction mix were added. The tubes were kept on ice for 30 min. Then, the cells were heat shocked at 42°C for 30 seconds and were immediately chilled on ice for 5 min. Then, 450 uL of prewarmed sterile LB medium was added. The tubes were incubated with shaking at 37°C for 1 hour. Lastly, 50 uL of the sample was plated on LB supplemented with kanamycin.

Colony PCR

After transformation, plates were checked the next day, and single colonies were picked using a disposable inoculation loop and washed in a PCR tube containing 10 uL of sterile water. Afterward, using the same loop, it was streaked on a “replica” plate containing LB and kanamycin. The plates were incubated at 37°C overnight. The cells in the PCR tubes were lysed at 95°C for 2 minutes using a thermocycler. Next, the debris was centrifuged at 2000 x g for 5 minutes. The supernatant was recovered and used as a template to perform a Taq polymerase PCR under the appropriate conditions. The right primers were used for the amplification and the length of the amplicon was checked using agarose gel. The samples were then sequenced following Macrogen Ez-Seq instructions.

Glycerol stock

Glycerol stock was prepared to store bacterial cultures for the long term. First, overnight liquid cultures were prepared using LB and adequate antibiotic resistance and incubated at 37°C overnight with shaking. After having bacterial growth, 900 uL of the liquid culture was added to 900 uL of 40% glycerol stock in a cryovials and mixed gently. The tubes were stored in –80 °C.

Golden Gate

We used Golden Gate assembly to simultaneously and directionally assemble the fragments of our vector, linker and blocks. We accounted for a 2:1 ratio of insert: backbone. The procedure was carried out as follows

Competent Cell Preparation:

The cells used in the transformation were chemically competent cells. First, 5 mL of overnight cultures of DH10B cells were prepared. Next, 2 mL of the bacterial culture (1/100 volume) was diluted into 200 mL of freshly prepared LB. It was incubated at 37°C until it reached OD600 = 0.4. The total culture was split into 4 falcon tubes (50 mL) and pelleted at 4000 x g for 15 minutes at 4°C. After discarding the supernatant, the cells were resuspended in total 50 mL cold MgCl₂ (12.5 mL of MgCl2 in each falcon tube). The resuspension was done by gently tapping the falcon tubes on the hand palm without pipetting up and down. The cells were centrifuged at 4000 x g for 15 minutes at 4°C. The cells were gently resuspended again using CaCl₂ in a total volume of 50 mL as previously done. Then, the tubes are placed on ice for 30 min and centrifuged again. Afterwards, the pellets resuspended in 2.5 mL of 85 mM CaCl₂, 15% glycerol. The content of the falcon tubes is combined and centrifuged. The cells are resuspended gently in 2 mL 85 mM CaCl₂, 15 % glycerol. Finally, a 50 uL aliquot was added into sterile 1.5 mL Eppendorf tubes while everything is kept on dry ice. The competent cells are stored in –80 °C.

Spot Assay

Overnight cultures of bacterial strains containing our expressed metal-binding proteins and control are prepared using 5 mL of LB with kanamycin. A gradient agar plate is prepared as follows [1]. In a square petri dish (12cm x 12cm), two batches of LB are poured. The first LB Batch (50 mL) is LB mixed with the highest metal concentration recommended by literature (usually 1000 mg/ L) and the second batch is LB with kanamycin and 0.02% of L-arabinose. The bottom of the square petri dish is marked at 0.9 cm from the bottom of the plate to indicate where the LB batch should reach. The metal-containing LB agar is poured first, tilting the dish to the mark, and allowing it to solidify before adding the LB layer.

After overnight incubation of the cultures, the optical density OD600 is measured and diluted to 0.2, and 0.02% arabinose is added to induce the expression. The cells are incubated again for 2- 3 hours. The OD is measured again, and the cultures are diluted to 10^6 cells/ mL at certain concentrations, and 3uL are plated onto the gradient agar plates. The latter are incubated overnight. The next day, the plates were scanned using the Typhoon™ FLA 9500 biomolecular imager at the correct wavelength to see the fluorescence of sfGFP expressed by the cells.

Toxicity Assay

Overnight cultures of bacterial strains containing our expressed metal-binding proteins and control are prepared using 5 mL of LB with kanamycin. The next day, the optical density is measured and diluted to 0.2. 0.02% of arabinose is added to induce the expression and the cultures are incubated for 2- 3 hours. Meanwhile, dilutions of the metal solutions to be tested are prepared: 10, 50, 75, 100, 250, 500, 750, and 1000 mg/L. After incubation is done, the optical density is checked again and converted to find the concentrations of cells. The latter is diluted until obtaining 1000 cells/ mL. A 96-well microplate is used where each well contains a total volume of 200 uL: 100 uL is the metal solution + 100 uL is the bacterial culture. Using the spectrophotometer, a reading is performed to collect the initial data. The microplate is incubated with shaking at 37°C with shaking. A second reading is performed after incubation.

Copper Colorimetric Assay

Similar to the toxicity assay, overnight cultures of bacterial strains containing our expressed metal-binding proteins and control are prepared using 5 mL of LB with kanamycin. The next day, the optical density is measured and diluted to 0.2. 0.02% of arabinose is added to induce the expression and the cultures are incubated for 3 hours. Meanwhile, dilutions of the metal solutions to be tested are prepared. In our colorimetric assay, we used three main reagents: ubiquinone, hydroxylamine hydrochloride, and acetate buffer. The biquinodone was prepared by mixing 0.02 g of biquinodone, and 100 uL of 100% triton-X in 50 mL of 70% ethanol. The sample is placed in a sonification match for 2 minutes and stirred with a magnetic stirrer at 40°C for 5 minutes. The Hydroxylamine Hydrochloride Solution was prepared by dissolving 0.75M of hydroxylamine hydrochloride in 20 mL of demineralized water. The acetate buffer is prepared by mixing 0.1 M of sodium acetate and 0.089 mL of acetic acid 99% in 50 mL of distilled water. pH is adjusted to 4.8 using 10N HCl.

After incubation, our cells are centrifuged at 9000 xg for 5 minutes. After discarding the supernatant, the cells are washed using 0.85% NaCl and centrifuged again. The cells are resuspended in 0.85% NaCl and the OD is measured to get the concentrations of cells. Using the 0.85% NaCl solution, cell culture, and metal stock solution, a dilution to reach 10^8 cells/mL and the correct concentration of the metal to be tested is performed. The samples are incubated for 1 hour and centrifuged again. The supernatant is collected. The samples are prepared as follows: 200 uL of supernatant, 200 uL of hydroxylamine hydrochloride, 1 mL of 2 2'-biquinoline, and 200 uL of acetate buffer. A blank is prepared too for comparison purposes: 200 uL of 0.85% NaCl, 200 uL of hydroxylamine hydrochloride, 1 mL of 2 2'-biquinoline, and 200 uL of acetate buffer. The tubes are centrifuged for 3 min at 11.5 xg. Finally, 250 uL of the supernatant is collected and plated on the 96-well microplate. The absorbance of the resulting solutions was measured using a spectrometer at 545 nm.

Cobalt Standard Curve Preparation

The standard curve preparation started by dissolving cobalt II chloride to prepare a main stock solution of 4000 mg/L. From the latter, we prepared 1.5 mL of eight standard solutions with the following concentrations: 10 mg/L, 25 mg/L, 50 mg/L, 100 mg/L, 150 mg/L, 200 mg/L, 250 mg/L, and 300 mg/L. For each standard, 200 uL of the solution is added to 80 uL of 6M hydrochloric acid, 200 ML of 50% potassium thiocyanate (KSCN), and 480 uL of acetone [2]. After thorough and careful mixing, the absorbance of each sample is measured using a spectrophotometer at 620 nm.

The absorbance values are recorded and plotted against the corresponding cobalt concentrations to create a standard curve. The latter is used as a reference for determining unknown cobalt concentrations by correlating their absorbance with the linear relationship established in the plot.