Team:ZJU-China/Protocols
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After the construction of the curli matrix and multi-enzyme complex, what we need to do next is to demonstrate the ability of the enzymes in a simple and intuitive way. An enzyme electrode sensor will be a good choice. The electrodes here can convert the biological signals into an electrical signal and transmit it to a portable instrument for us to use . In our design, by using the curli, gold nanoparticles and Nafion all together, we can amplify and finally collect the catalytic signal of the enzymes through the electrochemical way. In the following, we will explain one by one why every component in this design is necessary and how we use them to construct a well-run biosensor.
We obtained csgA sequence from the genome of E. Coli MG1655 by PCR and inserted it into Pet26(+). The recombinant plasmid was transfromed into BL21(DE3) and shaken overnight in LB with antibiotics. After reaching an optical density of 0.6~0.8 at 600 nm, the protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 25℃. We centrifuged the solution at 12000 rmp for 15mins, and cell pellets were resuspended gently with lysis buffer followed by sonication at the power of 300w for 30mins. The supernatant after centrifugation was kept for future purification.
Purification and Analysis Agarose Gel ElectrophoresisDissolve the agarose powder in TAE. Heat it to near-boiling point, cool sufficiently and pour the solution into a cast with a comb. After solidification, set gel tray into cuvette, filled with 1x TAE buffer. Load samples into the walls created by the comb. Run gel at 90-120V for 20-40min. Check image under UV light.
Ni-NTA Affinity PurificationCentrifuge the E. coli Lysates at 12000rmp at 4℃ for 20 minutes. Wash the column with 5ml sterile water, 8 mol/L urea, and sterile water. Add 0.1 mol/L Nickel Sulfate until the column changes from white to blue (Ni). Remove the excess Nickel with sterile water. Balance the column with wash solution. Add supernatant into the Ni-NTA column. Control the flow rate to about 0.5-1ml/min. Collect sample with EP tube.
SDS-PAGEGel production:Put two sealed glass plates together and clamp in a stand to make a mold. Mix H2O, 30%(Acr-Bise), gel buffer, SDS, TEMED and 10%APS to make a gel solution. Pour the solution into the mold without creating bubbles, and add water. After the polymerization of the separating gel, the water is discard and add the stacking gel solution which is made similarly.
Sample preparation: Add the sample buffer, and thus SDS in excess to the proteins, and the sample is then heated to 95℃ for five minutes.
Load 15-20 ul supernatant per lane on a protein gel. Load 10 ul prestained protein ladder. Run protein gel at 200V until dye front reaches the bottom of the gel.
Cango Red AssayAs an amyloid protein, we dyed curli by Cango Red to indicate the expression and characteristic of curli. Centrifuge curli pellet at 12,000g for 5 mins. Resuspend gently by LB and add Cango Red to 30μg/ml CR and incubate at RT for 30 mins. Centrifuge at 14,000rpm for 5mins. Set LB with equal CR as the blank. Measure the absorbance of supernatant at 480nm for CR and 600nm for cell concentration. Compare the expression in CsgA knockout Defect bacteria with that of our recombinant recombinant strain.
We use enzyme electrode to test enzymes’ activity and related electrodes’ response to substrates.
GOD-GCE• Modification
GCE (glassy carbon electrode) is cleaned ultrasonically and washed by water, alcohol, water in order. GCE is activated in PBS (pH=6.8) via CV method(cyclic voltammetry). Dissolve GOD (Glucose oxidase) in PBS (pH=6.8) to 10mg/mL. Add 10μl GOD solution to GCE’s working surface and dry it in 4℃. Add 5μL AuNP solution (disperse 5nm gold nano-particle with water to 5mg/mL) to GCE’s working surface and dry it in 4℃. Later, 5μL nafion (0.5wt% in water) is added in the same way. The GOD-GCE is preserved in PBS (pH=6.8) in 4℃.
• Test
A 3-electrode electrochemical cell (Pt electrode is used as counter electrode, Ag/AgCl electrode is used as reference electrode and GOD-GCE is used as working electrode.) is used for CV tests(The voltage scan rate is 50mV/s and scan range is 0.8V to -0.4V) and then to get amperometric I-t data (initial potential is 0.36V), and PBS (pH=6.8) with glucose’s concentrations varying from 1mM to 8mM is used as electrolyte solution.
HRP-GCE• Modification
GCE preparation is the same as GOD-GCE and is activated in PBS (pH=6.0) via CV method. Dissolve HRP (horseradish peroxidase) in PBS (pH=6.0) together with NADH (β-Nicotinamide adenine dinucleotide disodium salt) and both reach the concentration of 1mg/mL. Add 10μL HRP solution into GCE’s working surface and dry it in 4℃. Then GCE is modified with AuNP and nafion following same method above. The HRP-GCE is preserved in PBS (pH=6.0) in 4℃.
• Test
A 3-electrode electrochemical cell (Pt electrode is used as counter electrode, Ag/AgCl electrode is used as reference electrode and HRP-GCE is used as working electrode.) is used for CV tests (The voltage scan rate is 50mV/s and scan range is 1.5V to -1.0V.) and then to get amperometric I-t data (initial potential is -0.60V), and PBS (pH=6.0) with hydrogen peroxide’s concentrations varying from 1mM to 26mM is used as electrolyte solution.
LDH-GCE• Modification
GCE preparation is the same as GOD-GCE and is activated in PBS (pH=7.0) via CV method. Dissolve LDH (lactate dehydrogenase) in PBS (pH=7.0) to 1mg/mL. Add 10μL LDH solution to GCE’s working surface and dry it in 4℃. Modify GCE with AuNP and nafion following the method above. The LDH-GCE is preserved in PBS (pH=7.0) in 4℃.
• Test
A 3-electrode electrochemical cell (Pt electrode is used as counter electrode, Ag/AgCl electrode is used as reference electrode and LDH-GCE is used as working electrode.) is used for CV tests (The voltage scan rate is 50mV/s and scan range is 1.2V to -1.0V.) and then to get amperometric I-t data (initial potential is -0.45V), and PBS (pH=7.0) with lactic acid’s concentrations varying from 1mM to 10mM is used as electrolyte solution.
Enzyme electrode experiment used to test our logic gates StatementWe use this type of enzyme electrode to demonstrate that our idea about enzyme logic gates (XOR and AND) works.
XOR-GCE• Modification
GCE preparation is the same as GOD-GCE and is activated in PBS (pH=7.0) through CV method. Add 5μL 100nM Ni-NTA to GCE’s working face followed by 5μL CsgA solution (the curli part of our project) and 5μL 2% (m/v) glutaraldehyde and 2% (m/v) paraformaldehyde solution. After 2 hours in 4℃, wash the electrode with double distilled water and then dry it in room temperature. GOD-HRP-LDH complex is diluted in PBS (pH=7.0, with 1mg/mL NADH) and reach the concentration of 1mg/mL. Add 20μL of the solution mentioned above to GCE’s working surface and dry it in 4℃. Then modify GCE with AuNP and nafion following the methods above. The XOR-GCE is preserved in PBS (pH=7.0) in 4℃.
• Test
A 3-electrode electrochemical cell (Pt electrode is used as counter electrode, Ag/AgCl electrode is used as reference electrode and XOR-GCE is used as working electrode.) is used for CV tests (The voltage scan rate is 100mV/s and scan range is 2.0V to -1.2V.) and then to get amperometric I-t data (initial potential is -0.40V), and PBS (pH=7.0) with different concentration of glucose and lactic acid is used as electrolyte solution. In XOR gate, there are 2 inputs, glucose(G) and lactic acid(L), and totally 4 states, 0G-0L, 0G-1L, 1G-0L and 1G-1L. 0G (0L) means low concentration of glucose (lactic acid), which is 4mM. And 1G (1L) meas high concentration of glucose (lactic acid), which is 30mM.
AND-GCE• Modification
GCE preparation is the same as XOR-GCE and is activated in PBS (pH=6.8) via CV method. Follow the same method as XOR-GCE, modify GCE with Ni-NTA solution, CsgA solution, 2% (m/v) glutaraldehyde and 2% (m/v) paraformaldehyde solution, GOD-HRP complex in PBS (pH=6.8, with 1mg/mL NADH), AuNP and nafion. The AND-GCE is preserved in PBS (pH=7.0) in 4℃.
• Test
A 3-electrode electrochemical cell (Pt electrode is used as counter electrode, Ag/AgCl electrode is used as reference electrode and AND-GCE is used as working electrode.) is used for CV tests (The voltage scan rate is 100mV/s and scan range is 2.0V to -1.2V.) and then to get amperometric I-t data (initial potential is 0.6V), and PBS (pH=6.8) with different concentration of glucose and norepinephrine is used as electrolyte solution. In XOR gate, there are 2 inputs, glucose(G) and norepinephrine (N), and totally 4 states, 0G-0N, 0G-1N, 1G-0N and 1G-1N. 0G means low concentration of glucose, which is 4mM. And 1G means high concentration of glucose, which is 30mM. 0N means low concentration of norepinephrine, which is 2.2nM. And 1N means high concentration of norepinephrine, which is 3.5μM.
Detector - enzyme electrode used to detect one’s injury StatementWe designed an IDE (interdigital electrode, see Figure 1) and a 2D printer to print enzyme complex on it. Also, we made a set of hardware and software to detect and transmit current data. Through this data we can tell whether one is injured.
Modification
Paint Ag/AgCl ink on reference electrode of IDE (red part in Figure 1). Put IDE in oven, keep temperature at 120℃ for 5 minutes and cool it. Stick IDE inside the 2D printer and print Ni-NTA solution, CsgA solution and 2% (m/v) glutaraldehyde and 2% (m/v) paraformaldehyde solution. An hour later, wash the electrode in double distilled water and then dry it in room temperature. Stick IDE inside the 2D printer and print GOD-HRP-LDH complex solution used in XOR-GCE. After air-dry, print GOD-HRP-LDH complex solution again and dry IDE at room temperature. Print AuNP solution followed by air-dry. Print nafion and store IDE at room temperature for 4 days or at 4℃ for longer.
TestInsert IDE into our hardware, connect it with iPhone through Bluetooth. Add a drop of blood sample on IDE. The current shows whether the patient suffers from abdominal trauma, TBI (traumatic brain injury) or not.
• Mix 20 fmol (1 nM final concentration) backbone and 40 fmol each insert of target DNA segments together. The volume of this mixture must be 16 µL.
• Add water to a final volume of 16 µl
• Add 2 µL of 10× T4 DNA ligase buffer. Mix by vortexing.
• Add 1 µL of BsaI or BsmBI and 1 µL of T7 DNA ligase. Mix by gently pipetting.
• Incubate the reaction for 30 temperature cycles (42°C for 5 min and then 16°C for 5 min), followed by a final 10 min incubation at 55°C.
• Use 4 µLof this assembly reaction for transforming chemically competent cells.
After reaching an optical density of 0.6~0.8 at 600 nm, the protein expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 25℃. We centrifuged the solution at 12000 rmp for 10 mins, and cell pellets were resuspended gently with lysis buffer followed by sonication at the power of 300w 30mins. The supernatant after centrifugation was kept for future purification.
Purification and Analysis Agarose Gel ElectrophoresisDissolve the agarose powder in TAE. Heat it to near-boiling point, cool sufficiently and pour the solution into a cast with a comb. After solidification, set gel tray into cuvette, filled with 1x TAE buffer. Load samples into the walls created by the comb. Run gel at 90-120V for 20-40min. Check image under UV light.
Ni-NTA Affinity PurificationCentrifuge the E. coli Lysates at 12000rmp at 4℃ for 20 minutes. Wash the column with 5ml sterile water, 8 mol/L urea, and sterile water. Add 0.1 mol/L Nickel Sulfate until the column changes from white to blue (Ni). Remove the excess Nickel with sterile water. Balance the column with wash solution. Add supernatant into the Ni-NTA column. Control the flow rate to about 0.5-1ml/min. Collect sample with EP tube.
SDS-PAGEGel production:Put two sealed glass plates together and clamp in a stand to make a mold. Mix H2O, 30%(Acr-Bise), gel buffer, SDS, TEMED and 10%APS to make a gel solution. Pour the solution into the mold without creating bubbles, and add water. After the polymerization of the separating gel, the water is discard and add the stacking gel solution which is made similarly. Sample preparation: Add the sample buffer, and thus SDS in excess to the proteins, and the sample is then heated to 95℃ for five minutes. Load 15-20 ul supernatant per lane on a protein gel. Load 10 ul prestained protein ladder. Run protein gel at 200V until dye front reaches the bottom of the gel. Stain with coomassie blue for 1h and destain by destaining solution overnight.
Western blotIncubate the gel in Mouse-anti-His mAb for 1 hour at room temperature (or 37°C) with shaking. Wash in TBS three times 5 minutes each. Incubated the gel in alkaline phosphatase conjugated antibody against the primary antibody. Typical concentration is 1:1,000 in TBS. Add a small amount of BSA or NFM to act as carrier. Incubate for 1 hour at room temperature (or 37°C) with shaking. Wash in TBS three times 5 minutes each.
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