All experiments using Escherichia coli (E. coli) BL21 cells were grown in LB-Miller (Table 1, USBiological) for liquid cultures and LB-Miller agar (Table 1, chsProtocols) for solid plated bacteria. When carrying kanamycin resistance, 50 µg/mL kanamycin was added to both the liquid and solid cultures. In smaller cultures (1-25 mL), the shaking was set to 200 rpm. In large cultures (1 L), the shaking was set to 120 rpm. Small cultures were inoculated in 15- or 50 mL tubes, while larger ones in 3 L baffled flasks.
To enable transformation, 25 mL E. coli (BL21) cells were cultured for 6 hours at 37 °C with appropriate antibiotics (if already carrying plasmids) in a shaking incubator. Harvest of the cells was done by centrifugation for 5 minutes at 3000 g. The supernatant was discarded and the pellet was resuspended in 30 mL 0.1 M CaCl2 (Table 3). The centrifugation and resuspension were repeated once and afterward the cells were resuspended in 0.1 M CaCl2 with 15 % glycerol. Aliquots of the cells were made in a volume of 50 µL in 1.5 mL tubes. The now competent cells were placed directly into -80 °C, without prior use of liquid nitrogen.
The dry DNA from Integrated DNA Technologies was centrifuged briefly and resuspended in 50 µL autoclaved dH2O. A volume of 1 µL of the DNA solution was transferred into a tube containing 50 µL BL21 competent cells. This solution with bacterial cells and plasmid DNA was mixed by flicking the tube lightly. The tubes were incubated on ice for 30 min. Heatshock was carried out at 42 °C for 45 seconds and directly afterward, the tube was placed back on ice. The tubes were left on ice until they could be transferred into 1 mL LB-Miller media and incubated at 37°C for 1 hour. To plate these transformed bacteria, the culture was moved into a suitable tube and centrifuged at 500 g briefly. To concentrate the cells, 900 µL of the media was discarded, and the pelleted cells were resuspended in the remaining 100 µL. Plating was done on LB-Miller agar with 50 µg/mL kanamycin, all of the 100 µL was plated. The plates were then incubated at 37 °C until colonies had formed.
Plasmid preparation was done using Monarch® Plasmid DNA Miniprep Kit (New England Biolabs) protocol. A volume of 5 mL of LB media was added to a 50 mL tube together with suitable antibiotics. An inoculated colony was then added to the mixture and it was incubated overnight at 37 °C. The tubes were then centrifuged for 5 minutes at 3500 g. The supernatant was discarded and replaced with 200 µL Plasmid Resuspension Buffer (B1). The tubes were vortexed to ensure no clumps formed. The cells were then lysed by adding 200 µL Plasmid Lysis Buffer (B2). The tubes were inverted gently 5-6 times and then incubated for 1 minute. After this incubation, 400 µL of Plasmid Neutralization Buffer (B3) was added to the tubes. The tubes were inverted gently until the color changed to a uniform yellow. Tubes were then incubated for 2 minutes. The mixture was then centrifuged at 16000 g for 5 minutes. The supernatant was removed and placed in the spin column and centrifuged for 1 minute. Flowthrough was discarded. The spin-column was inserted in the collection tube and 200 µL of Plasmid Wash Buffer 1 was added and centrifuged for 1 minute. A volume of 400 µL of Plasmid Wash Buffer 2 was then added and centrifuged for 1 minute. The column was then transferred to a 1.5 mL microfuge tube and 50 µL autoclaved dH2O was added to the center of the matrix. The microfuge tubes were incubated for 1 minute, then spun for 1 minute to elute DNA. DNA concentration was measured using a Nanodrop. Samples were afterward loaded onto an agarose gel for gel electrophoresis to confirm the size of the plasmids.
Preparation of an agarose gel was done by mixing 1.3 g of agarose in 100 mL of TAE 1X buffer (Table 4). The mixture was microwaved until it boiled and then poured into a mold. Before the gel had hardened, a comb was added to form the wells. To prepare the samples to run on the gel, 7 µL of loading dye was mixed with 7 µL of each DNA sample. EDN, ECP, and HNMT were loaded, as well as 7 µL of a ladder for reference (1 kb plus DNA ladder, New England Biolabs). The gel was run at 100 V for 60 min. After finishing, it was stained with ethidium bromide for 5 min (with light shaking). To remove excess EtBr, destaining with TAE buffer was done for 1 minute (with light shaking). To analyze the results, the gel was placed in a gel camera (C150, Azure Biosystems).
Seeds were prepared by spreading 100 µL from a liquid culture of pIDTSmart-ECP/EDN E. coli (BL21) onto LB-Miller agar plates with 50 µg/mL kanamycin. A total of 3 plates were made for each construct. These plates were incubated at 37 °C for 16 hours. To inoculate the three plates, 400 µL of LB-Miller was poured onto them and released from the plate using a spreader. Cells from all the plates were then moved into a culture flask with 1 L 2X LB-Miller and 50 µg/mL kanamycin. This inoculation was then left to grow until an OD600 at 0.6 for both of the constructs. The induction of the T7 system was done with 1 mM IPTG and the temperature of the incubator was lowered to 20 °C. The cells were incubated for 16 hours. The optical density (600 nm) at harvest was measured to 5.7 for both of the constructs. Harvest was done by centrifugation at 3500 g 4 °C for 20 minutes using a swing-out rotor. The bacterial pellets were then resuspended using Buffer G (Table 5) and placed at -80 °C [1, prion protein section].
Bacteria lysis was done using a sonication method. Pellets of harvested cells were resuspended in the appropriate buffer and frozen at -80 °C. The cells were then thawed at 37 °C. A sonication probe was placed in the bacterial slurry and sonication was done at 30 % amplitude with 30 seconds ON 30 seconds OFF for a total ON time of 4 minutes. Samples were kept on ice during sonication to prevent excessive heating of the sample. After sonication samples were centrifuged at 10000 g for 40 minutes. The supernatant was saved for further purification.
IMAC purification can be used for His-tagged proteins. The protocol was adapted from [1, prion protein section]. A volume of 1 mL of Nickel-nitrilotriacetic acid agarose was added to a PD10 column and washed with 10 mL of wash buffer (Buffer B). The lysate was poured onto the IMAC column and flowthrough was saved until protein has been detected in the purified sample. A volume of 10 mL buffer G was added, followed by adding 7 mL of 50 % buffer G and 50 % buffer B (Table 5), and 7 mL buffer B afterward. Protein was eluted using 10 mL of buffer E (Table 5). All fractions were then run on the SDS-PAGE (Table 6) to verify the purity of the protein.
The separation gel was cast first, with the stacking gel directly after the separation gel had hardened (gel mold, Mini-Protean Tetra Casting Module, Bio-Rad). A comb (10 well 1 mm, 40 µL) was added when the stacking gel was cast.
To prepare samples to run on the SDS-PAGE gel, 20 µL sample was mixed with 18 µL sample buffer (Table 7) and 2 µL β-mercaptoethanol. All of this solution could then be loaded onto the gel.
The gel was placed in the gel electrophoresis tray and 1X running buffer (Table 8) was added (~0.5 L). Voltage was set to 100 V and the gel was run until all samples wandered into the separation gel. Then the voltage was then increased to 200 V and the gel was run until the sample buffer color front reached the bottom.
The gel was removed from the gel electrophoresis tray and rinsed with water twice, a final rinse was done with water and heated to near-boiling temperature. Following, the water was removed and the staining solution (Table 9) was added, this solution was heated to a near-boiling temperature as well. The gel was incubated with the staining solution for 30 minutes with light shaking. Afterward, the staining solution was removed and the destaining solution (Table 9) was added and left to destain for 30 minutes with a light shake. The destaining solution was changed once.
Protein dialysis was done in order to change the buffer conditions. A membrane (Standard RC tubing, MWCO 3.5 kDa, Spectrum Laboratories) was incubated in dH2O for 5 minutes and samples were loaded into the dialysis tubing. Dialysis was then done in 5 mM EDTA buffer (1 L) for 16 hours at 4 °C. The dialysis was repeated once but using a 5 mM borate buffer (1 L) instead.
To investigate the protein fold and stability, nanoDSF (Prometheus, NanoTemper) was performed. A volume of 10 µL of protein sample was loaded into capillaries (using capillary force) and simultaneously scanned at 330/350 nm wavelengths. The temperature was set to gradient and started at 20 °C, rising 0.5 °C/min until it reached 110 °C. For eosinophil cationic protein, 10 mM dithiothreitol was also added to investigate the state of the disulfide bonds.
ClusteRsy has revealed interesting biomarkers from the input data retrieved in the study GSE75011. These biomarkers and the biomarkers found from the literature study will need to be further validated to see that they indeed significantly over- or underexpressed in asthma patients. If found significantly differentially expressed they will move on to the next step where they will be expressed in bulk for the biosensor optimization. One idea we have for this step of phase II is also to create the antibodies used for the biosensor ourselves. This will be done by using a protocol for antibody production created by the Rochester iGEM team this year and then optimizing this protocol for our purpose (link). The biomarkers that are shown to be significant and have functionally expressed antibodies will finally make it to the biosensor assay.
Direct sandwich ELISA has been chosen as the method of validation due to its robustness and since all the biomarkers chosen have a matching pair of antibodies. The procedure of the validation will be as follows: 1. The capture antibodies are immobilized on the bottom of the wells. 2. The microplate is washed. 3. Our sample is added into the wells. 4. The microplate is washed. 5. The detection antibody with linked enzymes is added to their respective well. 6. The microplate is washed. 7. Enzyme substrate added to each well. 8. The reaction is noted. The amount of binding can now be determined.
Lateral flow assays (LFA) are simple and economic devices used as biosensors. During phase II of this project, an LFA will be designed that can detect the presence of overexpressed genes related to asthma, for which biomarkers were found using the software ClusteRsy. The development of ClusteRsy has been the focus of phase I of this project.
A LFA biosensor consists of several parts, but most important is the porous material designed to transport fluid and the test- and control lines where the detection of biomarkers occurs. At the test line, there are immobilized antibodies for the biomarkers. When they bind to the antibodies there is a colorimetric change, which is easy to detect for the person conducting the test. How this color change occurs depends on the type of LFA, it can either be a competitive assay or a sandwich assay. At the control line, there is a different antibody and if there is a colorimetric change there the test can be considered invalid.
For this project the idea is to design an LFA biosensor where there are several test lines for the different biomarkers. This can then be used to find which genes are overexpressed by the patient. This way the type of asthma the patient has can be detected which can then be used to determine the correct treatments. In order to accurately detect significantly overexpressed genes the sensitivity of the LFA will be adjusted to [2].
[1]. Sandberg A, Nyström S. Purification and fibrillation of recombinant human amyloid-β, prion protein, and tau under native conditions. In: Sigurdsson EM, Calero M, Gasset M, editors. Methods in Molecular Biology. New York, NY: Springer New York; 2018. p. 147–66.
[2]. Shuai Zhao et al. State of the art: Lateral flow assay (LFA) biosensor for on-site rapid detection. Chinese Chemical Letters. 2018;29(11):1567-1577. https://doi.org/10.1016/j.cclet.2017.12.008.
