1.1 LB Preparation 

 Material statistics:

Material Name	Quantity	Notes & Parameters
Agar powder	3g	Gelling agent
Yeast extract	5g	Sugar/starch source
Trypton	10g	Protein & nitrogen source
NaCl	10g	Adjusts salinity
Kanamycin	300μL	Used for verifying plasmid transfer success later

 Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Measuring cylinder	1	Volume 1000mL
Erlenmeyer flask	1	Volume 1000mL
Scale	1	Precision of 1mg
Spatula	1
Petri dish	20
Disinfecting pot	1

 

LB Preparation:  

1. Measure 700mL of ddH2O using the measuring cylinder 
2. Weigh 7 g of tryptone, 3.5 g of yeast extract and 7 g of NaCl using the scale & spatula.  
3. Add 7 g of tryptone, 3.5 g of yeast extract and 7 g of NaCl to ddH2O. This is the liquid LB broth.   
4. Gel Preparation:  
5. Measure 300mL of ddH2O using the measuring cylinder.  
6. Weigh out 3g of tryptone, 1.5g of yeast extract, 3g of NaCl and 3g of agar powder using the scale & spatula.  
7. Add 3g of tryptone, 1.5g of yeast extract, 3g of NaCl and 3g of agar powder to the 300mL of ddH2O. This is the LB gel which will be used for culturing the modified bacteria later.  
8. Disinfection & Verification Preparation 
9. Place both the broth LB and gel LB into a sterilizing pot under 121ºC for 2hr 
10. Distribute the 300mL LB gel evenly among the 20 Petri dishes.  
11. Add 300μL of kanamycin distributed equally across the Petri dishes. [1] 

 Note：

[1] The pET-28a plasmid which will be used as a vector for the target genes later in the procedure encodes a gene which offers kanamycin resistance. This helps verify transformation success, as the E.coli will only survive the kanamycin if they have successfully received the gene for kanamycin resistance.

 

 

 1.2 Plasmid Extraction 

 

Material statistics:

Material Name	Quantity	Notes & Parameters
LB containing e.coli	15mL(3mL/trial)	This was cultured 24hr prior to the experiment
SP1	≥1250μL (250μL/trial)
SP2	≥1250μL (250μL/trial)
SP3	≥1750μL(350μL/trial)
Buffer solution	≥2500μL(500μL/trial)
Wash solution	≥5mL(1mL/trial)
Elution buffer	≥375μL(75μL/trial)
Binding columns	10(2/trial)

  Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Centrifuge	1	Capacity for 8000rpm-12000rpm, holds 1.5mL microtubes
Pipette	≥1	Capacity between 10μL-100μL
Pipette	≥1	Capacity between 100μL-1000μL
Test tube	≥1	Capacity 15mL (adjust as needed; 3mL needed/trial)
EP tubes	5(1/trial)	3mL
EP tubes	10(2/trial)	1.5mL
Styrofoam box with ice inside	1	Enzyme/DNA storage

Experimental Procedure 

E.coli Preparation: 

1. Culture 3mL of E. coli over 24hrs 
2. Extract 1.5mL using pipette of in-solution E. coli and transfer to EP tube 
3. Place in centrifuge at 8000rpm for 1min 
4. Remove EP tube from centrifuge 
5. Remove supernatant from EP tube via pipette, leaving the e.coli deposit at the bottom [1] 
6. Repeat steps 2-5 for remaining 1.5mL 
7. Repeat steps 2-6 for all 5 EP tubes 

Lysis:  

8. Add 250μL of SP1 solution and mix [2] 
9. Add 250μL of SP2, then gently turn the EP tubes 2-4 times over 2-3min [3] 
10. Add 350μL of SP3, then gently turn the EP tubes 10-20 times. Strands of genetic material should be visible after this. [4] 

Purifying Target Plasmid:  

11. Place all EP tubes in centrifuge at 12000rpm for 10min. [5] 
12. Remove EP tubes from centrifuge. 
13. Prepare new EP tubes with binding columns. 
14. Add 500 μL buffer solution to EP tube containing the binding column  
15. Place the EP tube containing the binding column & buffer solution within the centrifuge at 12000rpm for 1min.  
16. Remove EP tubes from centrifuge.  
17. Dispose of the eluate.  
18. Extract the supernatant from EP tubes and transfer to binding column attached to EP tube. Do not disturb the e.coli pellet at the bottom of the previous EP tube. [6] 
19. Add 500μL wash solution to the binding column. [7] 
20. Place EP tube & binding column into centrifuge under 9000 rpm for 30sec.  
21. Discard the eluate.  
22. Repeat steps 19-21 twice for each sample of E.coli 
23. Repeat steps 19-22 for all 5 samples. 
24. Place the binding column into a clean 1.5ml EP tube and add 75μl of elution buffer to the adsorption film in the center of the binding column.[8] 
25. Place all EP tubes & binding columns into centrifuge under 9000 rpm for 1min 
26. Remove the binding column from the EP tube and store the elution buffer in the styrofoam ice box.  

 Note：

[1] This concentrates the e.coli into a pellet.  

[2] SP1 acts as a buffer, providing a suitable environment for the e.coli 

[3] SP2 lyses the e.coli, releasing the pET-28a plasmid 

[4] SP3 neutralizes SP2 (which is alkaline) 

[5] This separates the light pET-28a plasmids out from the heavier components of the e.coli cell.  

[6] The supernatant contains the target plasmids and the pellet contains lysed e.coli remains 

[7] The wash solution displaces unwanted pieces of genetic material without the His-tag 

[8] The elution buffer displaces the target plasmids from the binding column.  

 

1.3 Target Protein DNA PCR 

 

Material statistics:

Material Name	Quantity	Notes & Parameters
2X mix	50μL	Contains thermus aquaticus DNA polymerase, free BPs, Mg+ (sourced from Vazyme Biotech )
BD-tau F primer (10μm/μL)	1μL (1μL/trial)
BD-tau R primer (10μm/μL)	1μL (1μL/trial)
BD-tau template	1μL
Τ-tau F primer (10μm/μL)	1μL (1μL/trial)
T-tau R primer (10 μm/μL)	1μL (1μL/trial)
T-tau template	1μL
ddH2O	D+844μL(22μL/PCR trial, 400μL/template dilution, D for diluting primers, varies)

  Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
PCR Machine	1
Pipette	1	Capacity between 1μL-50μL
PCR tubes		Capacity 200μL
Styrofoam box with ice inside	1	Enzyme/DNA storage

 

System Preparation:  

1. Add a volume of ddH₂O 10 times as specified by the label on the microtube containing the primer into said microtube to dilute it to a concentration of 10μm/μL. This figure varies for each primer. [1] 
2. Add 400μL of ddH₂O into the microtubes containing 4μg of template for BD-tau/T-tau and mix.  
3. Add, in order, 22μL of ddH₂O, 25μL of 2X mix, 1μL of both the F and R primer solutions [2] prepared during the 1^st step for the template for BD-tau/T-tau, in addition to 1μL of the template itself to the microtube 
4. Repeat steps 1-3 for each both BD-tau and T-tau 
5. Repeat steps 1-4 for all 6 sets of BD-tau and Τ-tau 
6. Transfer all systems to a PCR machine 
7. PCR cycle:  
8. Preheat 95ºC for 5min [3] 
9. Heat to 95ºC for 15sec [4] 
10. Cool to 63ºC for 30sec [5] 
11. Heat to 72ºC for 1min [6] 
12. Repeat ii-iv 35 times 
13. Hold temperature at 72ºC for 10min [7] 
14. Cool to 12ºC for storage [8] 

 Note：

[1] Each primer is of different length and thus requires a different volume of solvent to dilute to the target molarity. The figure given on the tube is the volume of ddH2O necessary to dilute the primers to 100μm/μL.  

[2] These solutions containing the primers have a primer concentration of 10μM/μL 

[3] This denatures all strands of target DNA (breaks the H-bonds), allowing for primer attachment later. The temperature is held here to ensure thorough denaturing. 

[4] This temperature spike serves the same purpose as the 10-minute one and occurs for each cycle.  

[5] This allows for primer attachment. The temperature varies depending on the chosen primer. 

[6] This is the optimal functioning temperature for T. aquaticus polymerase, thus starting the replication process.  

[7] This ensures that the polymerization process is fully complete for all newly produced strands.  

[8] Setting the temperature lower stabilizes the newly replicated strands of DNA.  

1.4 Gel Electrophoresis 

 

Material statistics:

Material Name	Quantity	Notes & Parameters
1X TAE powder	1g (1g/1L of gel)	Working concentration 40mM Tris base, 20mM acetic acid, 1 mM EDTA, pH 8.0
ddH2O	100mL
Agarose powder	1g (1g/1L of gel)
10,000X YeaRed Nucleic Acid Gel stain	10μL(10μL/block of gel)
6X Loading buffer	20μL(10μL/trial)	Contains bisphenol blue
BD-tau PCR System	250 (50μL/trial)	See “Target Protein DNA PCR” for specifics
T-tau PCR System	250 (50μL/trial)	See “Target Protein DNA PCR” for specifics

   Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Agarose gel electrophoresis mold	1
Agarose gel electrophoresis mold comb	1	13 holes total
Pipette	1	Capacity 1μL-50μL
Gel electrophoresis machine	1	Gel block oriented horizontally; DNA runs parallel to the ground, distinct from SDS-PAGE setup
Measuring cylinder	1	Capacity ≥ 100mL
Conical flask	1	Capacity ≥ 100mL

 

Gel Block Preparation:  

1. Measure 1 L ddH₂O using the measuring cylinder and transfer to a conical flask.  
2. Add 1 bag of TAE buffer (~18.4 g) to the ddH₂O in step 1.
3. Add 1g of agarose powder to the ddH₂O in step 1.   
4. Mix the solution thoroughly by shaking the flask until all added powders are dissolved.  
5. Pour the solution into the gel electrophoresis mold.  
6. Place the comb into the gel electrophoresis mold and fix it in place in the designated slots. Wait for the gel to solidify.
7. System Preparation:  
8. Add 10 μL of 6X loading buffer to the PCR system from the previous procedure.  
9. Mix evenly.  
10. Repeat steps 7-8 for all PCR systems across all trials.  
11. Electrophoresis:  
12. Place the gel block into the gel electrophoresis machine. Ensure that the side of the gel block with holes is placed opposite to the cathode.
13. Add the 1X TAE buffer to the electrophoresis setu[
14. Using the pipette, add 20μL of 2k marker to the topmost hole. 
15. Add 50μL of the BD-tau/T-tau PCR system into the subsequent holes.  
16. Repeat step 13 for both PCR systems.  
17. Repeat steps 13-14 for all trials.  
18. Set the gel electrophoresis machine to 150 V and allow it to run for 20-30min. Stop once the bisphenol blue in the loading buffer has reached near the end of the gel block.  

 

1.5 DNA Recovery From Gel 

 

Material statistics:

Material Name	Quantity	Notes
Anhydrous ethanol	1500μL(500μL/trial)
B2 buffer	1800μL(600μL/trial)
Wash solution	4500μL(1500μL/trial)

  Equipment statistics:

Equipment Name	Quantity	Notes
Water Bath	1
Gel transilluminator	1
Scalpel	1
Pipette	≥1	Capacity between 10μL-100μL
Pipette	≥1	Capacity between 100μL-1000μL
Centrifuge	1

 

Preparation:  

1. Check whether there is sediment in Buffer B2 
2. Add 500μL of anhydrous ethanol to the wash solution 
3. Adjust the water bath pot to 50ºC  
4. Gel Liquification 
5. Place the block of gel under the gel transilluminator. [1] 
6. Cut out the gel block containing the target fragment from the agarose gel using the scalpel and weigh it. Record the mass 
7. Transfer the gel containing the target DNA to an EPtube. Dispose of the rest of the gel block.  
8. Add 600μL B2 buffer to the EP tube containing the target DNA to the agarose gel block and place the EP tube in a 50ºC water bath for 11 minutes. [2] 
9. Transfer the solution from step 7 into the binding column, centrifuge at 8000rpm for 30 sec, then discard the eluate. [3] 
10. DNA Extraction:  
11. Add 500μL wash solution, then centrifuge the EP-tube-binding-column complex at 9000rpm for 30 seconds. Discard the eluate. [4] 
12. Repeat step 9 once 
13. Centrifuge the empty binding column [5] at 9000rpm for 1 minute 
14. Place the binding column into a clean 1.5mL EP tube and add 30μl of elution buffer to the adsorption film in the center of the binding column.  
15. Allow the elution buffer to sit at room temperature for 1 minute 
16. Centrifuge the binding-column-test-tube complex at 9000rpm for 1 minute and preserve the DNA solution in the tube. [6] 

 Note：

[1] This shows where the target DNA is in the gel. The DNA fluoresces due to the nucleic acid gel stain added in the previous procedure.  

[2] This liquifies the gel.  

[3] The centrifugal force pushes the solution through the adsorption film in the binding columns, leaving the DNA attached to the film.  

[4] This washes the film and removes unwanted materials from the adsorption film.  

[5] The binding columns still contain the BD-tau/T-tau DNA.  

[6] This removes the DNA from the binding column and suspends it in the elution buffer.  

 

1.6 Purifying Gel Recovery Products 

Material statistics:

Material Name	Quantity per Set	Notes & Parameters
B2 buffer	600μL (200μL/trial)
Wash solution	4500μL (1500μL/trial)
Elution buffer	90μL (30μL/trial)

  Equipment statistics:

Equipment Name	Quantity	Notes
Water bath	1
Centrifuge	1	Can generate 8000rpm-9000rpm
Pipette	≥1	Capacity between 10μL-100μL
Pipette	≥1	Capacity between 100μL-1000μL

 

Preparation:  

1. Add 200μL of B2 buffer to microtube containing recovered DNA. Repeat for all 3 types of DNA subject to PCR.  
2. Place the microtube containing the buffer & recovered DNA in a water bath set to 55ºC for 5 minutes.  
3. Washing: 
4. Transfer the buffer solution & recovered DNA to a binding column attached to a test tube. Repeat for all 3 types of DNA subject to PCR.  
5. Place all microtubes into centrifuge at 8000rpm for 30sec.  
6. Add 500μL wash solution to the binding column. Repeat for all 3 types of DNA subject to PCR.  
7. Place all microtube-binding column complexes into centrifuge at 9000rpm for 30sec 
8. Remove all microtube-binding column complexes from centrifuge.  
9. Repeat steps 5-7 twice, then do steps 6&7 again once without adding more wash solution. 
10. Extracting target DNA:  
11. Add 30μL elution buffer to binding column.  
12. Allow the elution buffer to sit for 1 minute inside the binding column.  
13. Place all microtube-binding column complexes in the centrifuge at 9000rpm for 1min. Preserve the liquid in the microtube.  

 

1.7 Double Digestion 

 

Material statistics:

Material Name	Quantity	Notes & Parameters
10X Buffer	15 μL (5 μL/trial)
T-tau DNA	25 μL (25 μL/trial)	Recovered from gel in previous procedure
BD-Tau DNA	25 μL (25 μL/trial)	Recovered from gel in previous procedure
pET-28a	25 μL (25 μL/trial)	Recovered from gel in previous procedure
Hind III	3 μL(1μL/trial)	Hind III is an endonuclease that recognizes the following sequence: 5' A^AGCTT 3' [1]
Nhe I	3 μL(1μL/trial)	Nhe I is an endonuclease that recognizes the following sequence: 5' G^CTAGC 3'

 

  Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Micropipettes (P10, P20, P200)	1 set
Sterile pipette tips	1 box
Microtubes	3	1 per reaction + extra, capacity 200μL
Test tube rack	1
Microcentrifuge	1
Vortex mixer	1
Water bath	1	Maintain 37°C
Styrofoam box with ice inside	1	Enzyme/DNA storage
Pipette	1	Capacity between 1μL-20μL

 

System Preparation:  

1. Add 15 μL ddH₂O, 10μL of the target type of DNA for double digestion, 5μL of the 10X buffer, 1 μL Hind III and 1 μL Nhe I to a PCR tube.  
2. Repeat step 1 for both remaining types of DNA subject to double digestion (T-tau DNA/BD-Tau DNA/pET-28a).  
3. Place all samples into microcentrifuge for 30sec.  
4. Incubation:  
5. Place all samples into test tube rack.  
6. Keep these solutions in the water bath and allow to incubate for 2h at 37ºC.  

 

1.8 Ligation 

Material statistics:

Material Name	Quantity per Trial	Notes & Parameters
10X buffer	2μL
T4 ligase:	1μL
Cleaved BD-tau/T-tau DNA sequences	3 μL
Cleaved pET-28a	1μL
ddH2O	13μL
PCR tubes	3

Ligated Solution Preparation:  

1. Set the water bath to 50ºC and wait for its temperature to reach 50ºC.  
2. Place the PCR tubes containing the double digestion solution in the water bath set to 50ºC for 15min. [1] 
3. System Preparation: 
4. Add 13μL ddH₂O, 3μL of the target DNA sequence, 2μL of the 10X buffer and 1μL of pET-28a to an EP tube.[2]  
5. Repeat step 1 for the remaining sequences subject to ligation (BD-tau/T-tau).  
6. Place all PCR tubes in microcentrifuge for 30sec 
7. Incubate in PCR machine for 1 hour under 16ºC 

Note：

[1] “^” represents the site of cleavage.  

[2] This denatures Nhe I and Hind III.  

 

 

1.9 Heat shocking 

 

Material statistics:

Material Name	Quantity	Notes & Parameters
BL21 culture
DH5a culture
PET-28a solution (T-tau)	10μL (5μL/trial)
pET-28a solution (BD-tau)	10μL (5μL/trial)
LB broth	≥3600μL (900μL/sample)	The broth should be sterile and should not contain kanamycin.
CaCl2

 

  Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Cooler	1	Generates temperatures of -80ºC or lower
Water bath	1	Temperature range includes 42ºC
Petri dishes	≥6	Prepared during LB preparation step; 0.1% kanamycin

 

Storage:  

1. Store the competent E.coli in a fridge set to -80ºC.  
2. Sample Preparation:  
3. Add 5μL of pET-28a solution carrying the BD-tau/T-tau gene to the BL21/DH5α e.coli samples and gently mix.  
4. Repeat step 2 three additional times to generate a total of four samples, representing all possible combinations of one E. coli strain with one gene (BL21 + BD-tau, BL21 + T-tau, DH5α + BD-tau, DH5α + T-tau). [1] 
5. Heat Shocking & Culturing:  
6. Place the microtubes containing the e.coli samples into a 42ºC water bath for 45sec. [2] Quickly transfer the samples to an ice bath to cool.  
7. Add 900 μL of kanamycin-free LB broth each microtube. Mix evenly by repeatedly drawing in and dispensing the LB using the pipette.  
8. Once the e.coli-LB solutions are evenly mixed, centrifuge at 8000rpm for 5min. 
9. Remove 900 μL of supernatant from all microtubes.
10. Use the samples to inoculate petri dishes. Allow the E.coli colonies to develop for 12-16h at 37ºC.
11. Preparation for PCR:
12. Pick random colonies in the culture & split each sampled colony into 2 equal portions; one will be used for PCR later the other inoculate LB broth [3] which contains 0.1% kanamycin from the LB preparation step [4].

 Note:

[1] 2 strains were cultured to find a (relatively) optimal strain for expressing the target proteins.  

[2] This sudden change in heat increases membrane permeability & makes the e.coli cells competent.  

[3] The petri dishes will be used to verify transfer success later, and the e.coli in the broth will be used for protein expression.  

[4] The pET-28a plasmid which will be used as a vector for the target genes later in the procedure encodes a gene which offers kanamycin resistance. This helps verify transformation success, as only e.coli which have received the kanamycin-resistance gene encoded on the pET-28a plasmid will survive the kanamycin.  

 

 

2.0  PCR For Target Genes Inside E.coli 

 

Material statistics:

Material Name	Quantity per Trial	Notes & Parameters
Buffer 2X	50 μL (5μL /sample)
F-primer	2 μL (0.2μL/sample)
R-primer	2 μL (0.2μL/sample)
ddH2O	46 μL (4.6μL/sample)
BL21 e.coli carrying BD-tau DNA	N/A [1]	Engineered for superior protein-expressing capabilities
BL21 e.coli carrying T-tau DNA	N/A	Engineered for superior protein-expressing capabilities
DH5a e.coli carrying T-tau DNA	N/A	Engineered for superior transformation efficiency
DH5a e.coli carrying T-tau DNA	N/A	Engineered for superior transformation efficiency

   Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Cooler	1	Generates
Microtubes	16	4 for each combination of DNA and e.coli strain

 

Preparing the System:  

1. Add 4.6T μLddH₂O, 5.0T μLbuffer, 0.2T μL F primer and 0.2T μL R primer to a test tube, where T is the number of desired trials. In this experiment, T=10.
2. Separate the system equally into T 10 μL portions.  
3. Gently scrape e.coli colonies off the petri dishes used to cultivate e.coli after the heat-shocking procedure using a pipette[1] .
4. Transfer some colonies to the microtubes containing the PCR solution. Swap the pipette tip between each culture. [2] 
5. Repeat steps 4-5 for all cultures.  
6. PCR:  
7. Preheat to 95ºC for 5min 
8. Heat to 95ºC for 15sec 
9. Cool to 65ºC for 15sec 
10. Heat to 72ºC for 10sec  
11. Repeat steps 8-10 30 times 
12. Hold temperature at 72ºC for 10min 

 Note:

[1] The colonies were extracted by scraping the petri dishes with a pipette tip, as opposed to being added as a solution, thus making their volume negligible.  

[2] This prevents cross-contamination.

 

 

2.1 Monitoring E.coli Growth Curve 

Material statistics:

Material Name	Quantity	Notes & Parameters
LB	8mL (2mL/test tube)	Contains 50ng/mL K+ (kanamycin)

   Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Shaker	1	The shaker is sealed and conditioned
Timer	1	Optional, a clock may be used instead
Spectrophotometer	1	500μL cuvette recommended
Test tube	4	Capacity 15mL (2 samples/test tube)
Pipette	≥1	Capacity between 100μL-1000μL

 

 

Sample Preparation:  

1. Mingle 2mL LB (K+ with 50ng/ml concentration) with 50 μL E. coli cultured in the heat shocking step. Mix evenly
2. Place all tubes into 37˚C shaker. 
3. Observation:  
4. Return to record data for OD600 at different points for both proteins. (0.5, 1, 2, 4, 6, 8, 12 and 24h) [1] 
5. OD 600 Protocol:  
6. Remove the cuvette from the spectrophotometer & clean its exterior if needed.  
7. Add 500μL of sterile LB to the spectrophotometer cuvette.  
8. Place the cuvette back into the spectrophotometer 
9. Measure the OD 600 value of the blank. [2] 
10. Remove & empty the cuvette.   
11. Transfer the culture to the cuvette and measure the absorbance. 
12. Place the cuvette back into the spectrophotometer. 
13. Measure the OD-600 of the sample.  

• Repeat steps v-viii for all samples.  
• Note:

[1] OD600 is an approach which measures the light absorbance of bacterial or yeast cultures at 600nm wavelength using a spectrophotometer. As the curve requires infrequent, periodic measurements, this procedure can take place alongside other procedures.  

[2] This pares the spectrophotometer. 

 

2.2 Protein Extraction & Purification

 Material statistics:

Material Name	Quantity	Notes & Parameters
Non-denaturing lysis buffer	5mL
Wash solution	3mL (500μL/Wash)
Ice

  Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Centrifuge	1	Can generate 1000rpm--10000rpm
Sonicator	1
Beaker	1

 

Sonification Lysis Preparation:  

1. Centrifuge the tubes containing the E. coli culture at 6000rpm and 4˚C for 15 minutes to concentrate the E. coli into a pellet.  
2. Extract & discard the supernatant using a pipette.  
3. Add 20mL non-denaturing lysis buffer to the pellet. [1] 
4. Vortex the solution to mix thoroughly.  
5. Sonification Lysis:  
6. Uncap the test tube containing the E.coli culture and place it into a beaker containing ice. [2] 
7. Place the beaker on the sonicator’s platform with the test tube’s opening positioned directly under the needle and raise the platform until the needle is touching the solution. Ensure that the needle is not touching the test tube walls. [3] 
8. Sonicate at 300 W with 2 s on/2 s off cycles for 30 min.  
9. Remove the test tube from the sonicator once sonication is complete.  
10. Repeat steps 5-8 for T-tau.
11. Extracting Target Proteins:  
12. Centrifuge for 30 minutes at 10000 rpm at 4˚C.  
13. Extract the supernatant, transfer to an 15 mL test tube and place it on ice.
14. Extract 20 μL of the supernatant for SDS-PAGE later.  
15. Add 1 mL his-tag purification resin to an EP tube.
16. Centrifuge at 1000rpm at 4˚C for 10 seconds. Discard the supernatant. [4] 
17. Add 500 μL non-denaturing lysis buffer to the EP tube and centrifuge at 1000 rpm at 4˚C for 10 seconds. [5] 
18. Repeat step 15 2 more times.  
19. Add his-tag purification resin to test tubes containing BD-tau. Mix evenly.
20. Incubate for 60min at 4ºC on a shaker. 
21. Add 10mL of BD-tau supernatant and His-tag purification resin into large binding columns. [6] Keep the bottom capped.  
22. Allow protein solution containing BD-tau bound to his-tag purification resin to pass through the binding column. The resin beads should remain above the filter.
23. Add 500μL wash solution to the binding column and allow it to pass through the binding column film.  
24. Collect 20μL of the eluate for sampling later. Store the rest.  
25. Repeat steps 20-21 2 more times.  
26. Add 500μL elution buffer to the binding column and allow it to pass through the binding column film. 
27. Collect 20μL of the eluate for sampling later. Store the rest.  
28. Repeat step 23-24 3 more times.  
29. Collect the final eluate.  

Repeat steps 10-26 for T-tau
 Note:

[1] This suspends the e.coli in preparation for lysis 

[2] Sonication generates heat. The ice cools the sample to prevent heat-induced protein denaturation.  

[3] The needle and test tube would damage each other if in contact for extended periods during operation.  

[4] This leaves only the resin on the binding column film. The eluate is the storage solution for the resin.  

[5] The non-denaturing lysis buffer is used to wash the resin

[6] This binding column contains the his-tag purification resin beads and is larger than the binding columns used in the EP tubes.  

 

2.3 SDS-PAGE

 Material statistics:

Material Name	Quantity	Notes & Parameters
ddH2O
30% Acr-Bis
Gel buffer A	3.75 mL
Gel buffer B	2 mL
Mixing cups

 Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
SDS-PAGE	1

 

Sample Preparation:  

1. Add 10 μL protein sample and 10 μL 2X loading buffer to an EP tube.  
2. Heat the microtube to 95˚C for 5 minutes.
3. Casting SDS-PAGE Gel: 
4. Fix the gel casting cassette into the casting frames.  
5. Prepare the 15% resolving gel by adding 3.6ml ddH₂O, 7.5 mL 30% Acr-Bis, 3.75 mL Gel Buffer A, 0.15 mL 10% APS and 0.009 mL TEMED to a mixing cup.  
6. Pipette the resolving gel into the gel casting cassette.  
7. Add anhydrous ethanol to the gel casting cassette until the ethanol level reaches the height of the shorter plate 
8. Wait for 10-15min.  
9. Decant the ethanol.  
10. Prepare the 5% stacking gel by adding 1.33mL ddH₂O, 0.67 mL 30% Acr-Bis, 2 mL Gel Buffer B, 0.04 mL 10% APS and 0.004 mL TEMED to a mixing cup. 
11. Pipette the stacking gel into the gel casting cassette. 
12. Insert the comb into the gel casting cassette.  
13. Wait for 10-15min.  
14. Carefully remove the combs from the gel casting cassettes.  

Buffer Preparation:  

1. Prepare a conical flask with capacity 1 L or greater.  
2. Add 1 bag (approx. 18.4g) of tris-glycine SDS buffer into the conical flask.  
3. Measure 1 L of water using a measuring cylinder.  
4. Add the water to the conical flask. 
5. Mix evenly until tris-glycine SDS buffer is fully dissolved.  
6. SDS-PAGE Electrophoresis:  
7. Transfer the cassettes to an SDS-PAGE gel electrophoresis tub.  
8. Add to each gel block the following samples:  
9. Markers for the first 2 cells. For the remaining 9 cells, add 10 μL CL, FT, W1, W2, W3, E1, E2, E3 and E4 in order.
10. Add buffer to the space between the cassettes within the tank until the space is completely full.  
11. Place the cap onto the setup. Ensure that the anodes and cathodes are properly paired.  
12. Plug the cap’s wires into an electrophoresis power supply. Ensure that the anodes and cathodes are properly paired. 
13. Turn on the power supply. Set the voltage to 60 V for 35min. Then, set the voltage to 130 V for 40min.  
14. Gel Dyeing:  
15. Cut the stacking gel from the gel block using a scalpel and discard it.  
16. Place the resolving gel block into a dyeing tub and add 20 mL of Coomassie brilliant blue dye to the tub.  
17. Place the dyeing tub onto a shaker and allow to dye overnight.  
18. Place the dyed gel block into an SDS-PAGE gel reader and document the results.  

 

3.1 SELEX screening for nucleic acid aptamers 

Material statistics:

Material Name	Quantity	Notes & Parameters
DNA Oligo Library	/	1503.75 μM (This will vary based on library)
ddH2O	/	/
PBS	/	/
Streptavidin magnetic bead solution	/	/
EP tubes	/	/
NaOH	/	/
T-tau	1.5ml	5mg/ml
BD-tau	1.5ml	5mg/ml
Ni-NTA beads	2ml

 Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Pipette	/	/
Microcentrifuge	1	1
Shaker	1	1
Magnetic separation rack	1	1
Nanodrop	1	1

SELEX screening for Table 1:

	{1}	{2}	{3}	{4}	{5}	{6}	{7}
Round	DNA Library (Concentration, Volume)	Counter-selection (Bead Volume, Time)	Positive BD-tau Selection (Bead Volume, Time)	Elution Volume	Secondary T-tau Counter-selection (Bead Volume, Time)	Wash (Volume, Repetitions)	PCR Cycles
1	40 µM, 500 µL	–	25 µL × 2, 60 min	100 µL	–	500 µL × 2	20
2	300 nM, 500 µL	–	25 µL × 2, 45 min	300 µL	–	500 µL × 2	14
3	300 nM, 500 µL	–	25 µL × 2, 30 min	300 µL	–	500 µL × 2	14
4	250 nM, 500 µL	–	50 µL × 2, 30 min	300 µL	–	500 µL × 2	14
5	250 nM, 500 µL	–	50 µL × 2, 30 min	300 µL	–	500 µL × 3	12
6	250 nM, 500 µL	–	50 µL × 2, 30 min	500 µL	400 µL, 30 min	500 µL × 3	12
7	250 nM, 500 µL	–	50 µL × 2, 30 min	500 µL	400 µL, 30 min	1000 µL × 3	22
8	250 nM, 300 µL	125–1000µL, 30 min	50 µL × 2, 30 min	300 µL	400 µL, 30 min	1000 µL × 3	26
9	250 nM, 300 µL	400 µL, 30 min	50 µL × 2, 30 min	300 µL	400 µL, 30 min	1000 µL × 3	24
10	250 nM, 300 µL	1000 µL, 30 min	50 µL × 2, 30 min	300 µL	1000 µL, 30 min	1000 µL × 3	10
11	250 nM, 300 µL	1000 µL, 30 min	50 µL × 2, 30 min	300 µL	1000 µL, 30 min	1000 µL × 3	18
12	250 nM, 300 µL	1000 µL, 30 min	50 µL × 2, 30 min	500 µL	1000 µL, 30 min	1000 µL × 3	16
13	250 nM, 300 µL	1000 µL, 30 min	50 µL × 2, 30 min	500 µL	1000 µL, 30 min	1000 µL × 3	16
14	250 nM, 300 µL	1000 µL, 30 min	50 µL × 2, 30 min	500 µL	1000 µL, 30 min	1000 µL × 5	20
15	250 nM, 300 µL	1000 µL, 30 min	50 µL × 2, 30 min	500 µL	1000 µL, 30 min	1000 µL × 5	14
16	100 nM, 200 µL	200 µL, 30 min	50 µL × 2, 30 min	500 µL	200 µL, 30 min	1000 µL × 5	12

Ni-NTA Bead Preparation:  

1. Thoroughly resuspend the stored Ni-NTA beads by gently shaking the bottle and pipetting the storage solution.  
2. Extract 20 μL Ni-NTA beads and transfer to a 1.5ml EP tube. 
3. Add 500 μL PBS and vortex thoroughly. Use microcentrifuge -10000rpm for 10 seconds.  
4. Place the microtubes in the magnetic separation rack and allow the beads to collect against the tube wall.
5. Carefully discard the supernatant without disturbing the beads. 
6. Repeat steps 4-6 3 times for each tube. For efficiency’s sake, sufficient beads for all subsequent rounds were washed at once.  
7. Bead Suspension & Incubation:  
8. Suspend the beads in 100 μL of PBS after washing.  
9. Add His-tag peptides at a concentration of 400 µg/mL for rounds 1–7, or 800 µg/mL for rounds 8–17.  
10. Incubate the tubes by placing them in a 4˚C shaker for 16-18hr. Maintain 4ºC for storage. 
11. DNA Library Preparation:  
12. Heat the library to 95˚C for 3 minutes 
13. Snap-cool for 5 minutes to form ssDNA.  

SELEX contains 16 rounds of selection, divided into Positive Selection (Rounds 1-5), Denoising I (Rounds 6-7) and Denoising II (Rounds 8-16). For each section, refer to its corresponding procedure. {n} is used to represent variable values. Refer to the corresponding cell in Table 14.1 at coordinates (n, round number) for the specific value of a given trial.

Positive Selection (Rounds 1-5):  

1. Add {1} nM of DNA library and {3} μL of BD-tau Ni-NTA magnetic beads to ({1(volume)} - - {3}) μL of PBS buffer in an EP tube. For round 1, Calculate with the following equation: , where is the volume of the library needed, is the molar concentration of the library and is the desired concentration.
2. Allow library &BD-tau Ni-NTA magnetic beads to incubate for Table1 column {3} min.
3. Add Table1 column {6} μL ddH2O to the EP tubes containing the library and Ni-NTA magnetic beads.
4. Place the EP tubes into the magnetic separation rack. Carefully discard the supernatant without disturbing the BD-tau Ni-NTA magnetic beads.
5. Repeat steps 3-4 an additional Table1 column {6} times.
6. Add Table1 column {4} μL Dnase-free ddH2O to the EP tube. Heat the supernatant to 95˚C for 7 minutes.
7. Place the system in the microcentrifuge for 30sec.
8. Conduct {7} rounds of PCR on the sample.
9. 20μL streptavidin magnetic beads separation of ssDNA
10. Conduct a nanodrop test for the supernatant to determine its concentration. 
11. Calculate for the next trial given by the following equation: , where is the volume of the library needed, is the concentration measured via nanodrop and is the desired concentration.

Denoising I (Rounds 6-7) 

1. Add Table1 column{1} nM of DNA library and Table1 column{3} μL of BD-tau Ni-NTA magnetic beads to Table1 column ({1(volume)} - - {3}) μL of PBS buffer to an EP tube.  
2. Allow the library & BD-tau Ni-NTA magnetic beads to incubate for Table1 column {3} min.
3. Add Table1 column {6} μL ddH₂O to the EP tubes containing the library and BD-tau Ni-NTA magnetic beads.
4. Place the EP tubes into the magnetic separation rack. Carefully discard the supernatant without disturbing the BD-tau Ni-NTA magnetic beads.
5. Repeat steps 3-4 an additional Table1 column {6} times.
6. Add Table1 column {4} μL PBS to the EP tube. Heat the supernatant to 95˚C for 7 minutes.
7. Place the test tube on the magnetic separation rack.
8. Transfer all eluted supernatant and Table1 column {5} μL of Ni-NTA magnetic beadsbeads to a new test tube.
9. Allow the beads and library to incubate for 30min.
10. Place the test tube on the magnetic separation rack. Extract all supernatant and transfer to a new EP tube.
11. Heat the supernatant to 95˚C for 7 minutes. Place the system in the microcentrifuge for 30sec.
12. Conduct Table1 column {7} rounds of PCR on the sample.
13. 20μL streptavidin magnetic beads separation of ssDNA
14. Add 500 μL ΝaΟΗ to the EP tubes to wash out dsDNA.
15. Conduct a nanodrop test for the supernatant to determine its concentration. 
16. Calculate for the next trial given by the following equation: , where is the volume of the library needed, is the concentration measured via nanodrop and is the desired concentration.

Denoising II (Rounds 8-16) 

1. Add Table 1 column {1} nM of DNA library and Table1 column {2} μL of unloaded BD-tau Ni-NTA magnetic beads to Table1 column ({1(volume)} - - {2}) μL of PBS buffer to an EP tube.  
2. Allow the library & BD-tau Ni-NTA magnetic beads to incubate for Table1 column {2} min.
3. Extract the supernatant and transfer to a new EP tube.
4. Add extract the supernatant to Table1 column {3} μL of BD-tau Ni-NTA magnetic beads.
5. Allow the library & BD-tau Ni-NTA magnetic beads to incubate for Table1 column {3} min.
6. Add Table1 column {6} μL PBS to the EP tube containing the library and BD-tau Ni-NTA magnetic beads.
7. Place the microtubes into the magnetic separation rack. Carefully discard the supernatant without disturbing the BD-tau Ni-NTA magnetic beads.
8. Repeat steps 4-5 an additional Table1 column {6} times.
9. Add Table1 column {4} μL PBS to the EP tube. Heat the supernatant to 95˚C for 7 minutes.
10. Place the test tube on the magnetic separation rack.
11. Transfer all eluted supernatant and Table1 column {5} μL of T-tau Ni-NTA magnetic beads to a new test tube.
12. Allow the beads and library to incubate for 30min.
13. Place the test tube on the magnetic separation rack. Extract all supernatant and transfer to a new EP tube.
14. Heat the supernatant to 95˚C for 7 minutes.
15. Place the system in the microcentrifuge for 30sec.
16. Conduct {7} rounds of PCR on the sample.
17. Conduct a nanodrop test for the supernatant to determine its concentration. 
18. Calculate for the next trial given by the following equation: , where is the volume of the library needed, is the concentration measured via nanodrop and is the desired concentration.

Note:

Prepare a 50 μL system for PCR:

Prime Star 2X buffer 25 μL, SELEX F&R Primer 1 μL+1 μL, Library 23 μL.

Step	Temp	Time	# of cycles
Initial Denaturation	95°C	90s
Denaturation	95°C	30 sec	Table 1
Primer Annealing	57°C	30 sec
Extension	72°C	60 sec
Final Extension	72°C	3 min

3.2 Flow Cytometry 

Material statistics:

Material Name	Quantity	Notes & Parameters
1X phosphate-buffered saline solution	/	/
SELEX samples for T-tau and BD-tau from rounds 1, 4, 7 and 14	80μL total (20μL/sample)	/

Equipment statistics:

Equipment Name	Quantity
Test tube	5
Cap sieve	5
Flow cytometer	1

 

 Process :

1. Add 2500μL phosphate-buffered saline solution to the extra test tube if needed.
2. Label a test tube with its respective trial number.
3. Gradually add 500μL of phosphate-buffered saline solution along with the PCR solution of the given SELEX cycle to the test tube designated for said round of SELEX. To ensure full transfer of the PCR solution, add some volume of phosphate-buffered saline solution to the PCR tube containing the SELEX library, repeatedly pipette the mixture, then extract it and transfer to the larger test tube. [1] 
4. Repeat for all cycles and for 2 control samples containing unbound streptavidin magnetic beads and unbound aptamers respectively. For this experiment, the library from the 7th SELEX cycle was diluted to 2 separate concentrations to find an optimal concentration for the cytometry machine later in the procedure.  
5. Pass all solutions through separate 35μm-pore-size sieves to prevent the sample from jamming the flow cytometry machine.  
6. Add a control/blank solution of unbound streptavidin magnetic beads to the cytometry machine to test it. 
7. Adjust the parameters of the flow cytometry machine:  
8. Set a range of detection. This will appear as a polygon on Graph 1 whose vertices may be determined by clicking on their desired locations. All datapoints within this polygon are registered.
9. Adjust the voltage until a clustered, elliptical distribution starts showing on Graph 1. The voltage used was ~0.62 V.
10. Export all data. Repeat for all trials.

Graph 1 shows the size & structural complexity of the aptamer-bead complexes along the Χ and Y axes respectively.

Graph 2 uses data and axes identical to graph one, but accounts for chimeric formations of the aptamers. 

Graph 3 shows the number of particles (Y-axis) fluorescing at a given intensity (X-axis) 

Table 2:

Graph Name	Contents & Parameters
(Graph 1)	X-coordinate equals particle size, Y-coordinate shows particle complexity.
(Graph 2	X-coordinate equals particle size, Y-coordinate shows particle complexity, accounts for chimeric formations
(Graph 3)	Shows number of particles fluorescing at a given intensity. X-axis represents different intensities, Y-axis represents number of particles.

3.3 SPR( Surface Plasmon Resonance)

A. Chip Surface Preparation and Immobilization

1. Obtain 5 mg/ml of the biotinylated target protein BD-tau from step 2.2.
2. Select a pre-coated streptavidin (SA) SPR chip (such as a CM5 chip).
3. Inject the biotinylated target protein BD-tau into a specific flow cell of the SA chip to allow specific and firm capture.

B. Binding and Dissociation Experiment

1. Dilute the nucleic acid aptamer with running buffer to concentrations of 0 μM, 5 μM, 10 μM, 30 μM, 100 μM, 100 μM, and 150 μM.
2. Inject the series of diluted aptamer solutions in ascending order of concentration over the chip surface immobilized with the target protein at a constant flow rate, monitoring the binding process.
3. After injecting each concentration sample for a period (typically 1–5 minutes), switch to running buffer to monitor the dissociation process (typically 5–10 minutes).
4. Between each cycle, briefly inject (30–60 seconds) a regeneration solution (for protein-nucleic acid interactions, 1–5 M NaCl) to thoroughly wash away the bound aptamer and regenerate the chip surface.

3.4 ELISA (Enzyme-Linked ImmunoSorbent Assay)

Material statistics:

Material Name	Notes & Parameters
BD-tau aptamer solution	5 mg/mL
T-tau aptamer solution	5 mg/mL
BSA solution	2mg/ml
Salmine
1XTBST	45mL of Distilled water, 5 mL of TBS, 25μL of Tris buffer salt solution containing Tween 20
Sealing buffer	/
BD-tau protein solution	5 mg/mL
T-tau protein solution	5 mg/mL
Streptavidin/Poly HRP
Aluminum foil	/
Synthetic blood	Contains H2O, important proteins, various electrolytes, O2, CO2, steroids, clotting agents, glucose, etc.

Equipment statistics:

Equipment Name	Quantity
ELISA board	10
Pipette	500
Scissors	1
Microplate reader	1

Binding Aptamers to Plate:

{1}	{2}	{3}
Aptamer type bound to ELISA board	Introduced Solution	Number of Cells
BD-tau	Polyprotein	60
BD-tau	T-tau	96
BD-tau	BSA	96
T-tau	BD-tau	93
BD-tau	BD-tau solution + polyprotein + BSA	96

 

1. Dilute the {1} aptamer solution with the coating solution (pH9.6) to 1μg/mL. 
2. Add 100μL of the diluted aptamer solution to each well of the 96-well plate. Incubate overnight at 4°C.
3. Blocking & Washing:  
4. Wash the plate 3 times by adding 200 μL 1X TBST to each well.
5. Add 200μL of sealing buffer to each well; Incubate at room temperature for 1 hour or at 37 ° C for 30 minutes. 
6. Wash the plate 3 times by adding 200 μL 1X TBST to each well.
7. Sample Preparation & Addition:  
8. Prepare standard curve {2} samples with concentrations 1, 2, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000 and 10,000 pg/mL.
9. Randomly generate 2 equal-sized sets of {2} sample concentrations, one below 100 pg/mL and the other above 100 pg/mL and below 10 ng/mL. (See Attachment 1 for c++ code for generating random concentrations).
10. Prepare H2O-based and artificial blood-based storage solutions containing {2}. The proteins are sourced from earlier in the procedure.
11. Prepare solutions of all randomly-generated concentrations. Use the H2O storage solution to prepare 25% of randomized sample concentrations and artificial blood for the remaining 75%.
12. Add 100 μL/well of standard curve protein solutions prepared earlier to the first 13 wells in the ELISA plate.
13. Add the remaining randomized samples prepared in step 9 to the subsequent wells. The number of samples may be adjusted. Reference {3} for the figures in the original experiment. Record the sample concentration in each well.
14. Incubation:  
15. Incubate at room temperature for 2 hours (or overnight at 4 ° C). 
16. Wash the plate three times with TBST.  
17. Fittest incubation:  
18. Add 100μL of biotin solution (500nM TBST+20 mg/mL biotin in PVP buffer+100 μg/mL salmine DNA) to each well. [1]
19. Incubate the plate at room temperature for 1 hour.  
20. Signal Detection:  
21. Wash the plate three times with TBST. 
22. Incubation of enzyme-labeled secondary antibodies  
23. Add 100 μL of Streptavidin Poly HRP to each well diluted at a ratio of 1:10,000 to TBST. [2]
24. Incubate at room temperature for 1 hour. Wrap the sample in aluminium foil.  [3]
25. Post-Incubation Washing:  
26. Wash the board 5 times with TBST. [4]`
27. Color Development & Measurement:  
28. Add 100μl of TMB chromogenic substrate to each well and react in the dark for 10-15 minutes. 
29. Add 50μL/well of termination solution with concentration 2M.
30. Measure the absorbance at 450nm using an microplate reader (reference wavelength: 450-620nm).  
31. Repeat the entire procedure for all 5 combinations of aptamers and introduced solutions.

Note: 

[1] This helps the biotin attach to the aptamer

[2] This helps the streptavidin poly HRP attach to the biotin. The streptavidin poly HRP has a peak absorbance rating of ~450 nm, which will be measured using abosorbance in step 22.

[3] This prevents light-induced damage to the dyes.

[4] This reduces noise by washing residual proteins away.

strong>1 Extract the pET28a plasmid

Material statistics:

Material Name	Quantity	Notes & Parameters
LB containing e.coli(pET28a)	15mL(3mL/trial)	This was cultured 24hr prior to the experiment
SP1	≥1250μL (250μL/trial)
SP2	≥1250μL (250μL/trial)
SP3	≥1750μL(350μL/trial)
Buffer solution	≥2500μL(500μL/trial)
Wash solution	≥5mL(1mL/trial)
Elution buffer	≥375μL(75μL/trial)
Binding columns	10(2/trial)

  Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Centrifuge	1	Capacity for 8000rpm-12000rpm, holds 1.5mL microtubes
Pipette	≥1	Capacity between 10μL-100μL
Pipette	≥1	Capacity between 100μL-1000μL
Test tube	≥1	Capacity 15mL (adjust as needed; 3mL needed/trial)
EP tubes	5(1/trial)	3mL
EP tubes	10(2/trial)	1.5mL
Styrofoam box with ice inside	1	Enzyme/DNA storage

Experimental Procedure 

E.coli Preparation: 

1. Culture 3mL of E. coli pET28a over 24hrs 
2. Extract 1.5mL using pipette of in-solution E. coli and transfer to EP tube 
3. Place in centrifuge at 8000rpm for 1min 
4. Remove EP tube from centrifuge 
5. Remove supernatant from EP tube via pipette, leaving the e.coli deposit at the bottom [1] 
6. Repeat steps 2-5 for remaining 1.5mL 
7. Repeat steps 2-6 for all 5 EP tubes 

Lysis:  

1. Add 250μL of SP1 solution and mix [2] 
2. Add 250μL of SP2, then gently turn the EP tubes 2-4 times over 2-3min [3] 
3. Add 350μL of SP3, then gently turn the EP tubes 10-20 times. Strands of genetic material should be visible after this. [4] 

Purifying Target Plasmid:  

1. Place all EP tubes in centrifuge at 12000rpm for 10min. [5] 
2. Remove EP tubes from centrifuge. 
3. Prepare new EP tubes with binding columns. 
4. Add 500 μL buffer solution to EP tube containing the binding column  
5. Place the EP tube containing the binding column & buffer solution within the centrifuge at 12000rpm for 1min.  
6. Remove EP tubes from centrifuge.  
7. Dispose of the eluate.  
8. Extract the supernatant from EP tubes and transfer to binding column attached to EP tube. Do not disturb the e.coli pellet at the bottom of the previous EP tube. [6] 
9. Add 500μL wash solution to the binding column. [7] 
10. Place EP tube & binding column into centrifuge under 9000 rpm for 30sec.  
11. Discard the eluate.  
12. Repeat steps 19-21 twice for each sample of E.coli 
13. Repeat steps 19-22 for all 5 samples. 
14. Place the binding column into a clean 1.5ml EP tube and add 75μl of elution buffer to the adsorption film in the center of the binding column.[8] 
15. Place all EP tubes & binding columns into centrifuge under 9000 rpm for 1min 
16. Remove the binding column from the EP tube and store the elution buffer in the styrofoam ice box.  

 Note：

[1] This concentrates the e.coli into a pellet.  

[2] SP1 acts as a buffer, providing a suitable environment for the e.coli 

[3] SP2 lyses the e.coli, releasing the pET-28a plasmid 

[4] SP3 neutralizes SP2 (which is alkaline) 

[5] This separates the light pET-28a plasmids out from the heavier components of the e.coli cell.  

[6] The supernatant contains the target plasmids and the pellet contains lysed e.coli remains 

[7] The wash solution displaces unwanted pieces of genetic material without the His-tag 

[8] The elution buffer displaces the target plasmids from the binding column.  

 

 

1.2 Target Protein DNA PCR 

 

Material statistics:

Material Name	Quantity	Notes & Parameters
2X mix	50μL	Contains thermus aquaticus DNA polymerase, free BPs, Mg+ (sourced from Vazyme Biotech )
Cas12a F primer (10μm/μL)	5μL (1μL/trial)
Cas12a R primer (10μm/μL)	5μL (1μL/trial)
Cas12a template(10μm/μL)	5 μL
ddH2O	D+844μL(22μL/PCR trial, 400μL/template dilution, D for diluting primers, varies)

  Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
PCR Machine	1
Pipette	1	Capacity between 1μL-50μL
PCR tubes		Capacity 200μL
Styrofoam box with ice inside	1	Enzyme/DNA storage

 

System Preparation:  

1. Add a volume of ddH₂O 10 times as specified by the label on the microtube containing the primer into said microtube to dilute it to a concentration of 10μm/μL. This figure varies for each primer. [1] 
2. Add 400μL of ddH₂O into the microtubes containing 4μg of template for BD-tau/T-tau and mix.  
3. Add, in order, 22μL of ddH₂O, 25μL of 2X mix, 1μL of both the F and R primer solutions [2] prepared during the 1^st step for the template for BD-tau/T-tau, in addition to 1μL of the template itself to the microtube.
4. Repeat steps 1-3 for each both BD-tau and T-tau 
5. Repeat steps 1-4 for all 6 sets of BD-tau and Τ-tau 
6. Transfer all systems to a PCR machine 
7. PCR cycle:  
8. Preheat 95ºC for 5min [3] 
9. Heat to 95ºC for 15sec [4] 
10. Cool to 63ºC for 30sec [5] 
11. Heat to 72ºC for 1min [6] 
12. Repeat ii-iv 35 times 
13. Hold temperature at 72ºC for 10min [7] 
14. Cool to 12ºC for storage [8] 

 Note：

[1] Each primer is of different length and thus requires a different volume of solvent to dilute to the target molarity. The figure given on the tube is the volume of ddH2O necessary to dilute the primers to 100μm/μL.  

[2] These solutions containing the primers have a primer concentration of 10μM/μL 

[3] This denatures all strands of target DNA (breaks the H-bonds), allowing for primer attachment later. The temperature is held here to ensure thorough denaturing. 

[4] This temperature spike serves the same purpose as the 10-minute one and occurs for each cycle.  

[5] This allows for primer attachment. The temperature varies depending on the chosen primer. 

[6] This is the optimal functioning temperature for T. aquaticus polymerase, thus starting the replication process.  

[7] This ensures that the polymerization process is fully complete for all newly produced strands.  

[8] Setting the temperature lower stabilizes the newly replicated strands of DNA.  

 

 

1.3 Gel Electrophoresis 

 

Material statistics:

Material Name	Quantity	Notes & Parameters
1X TAE powder	1g (1g/1L of gel)	Working concentration 40mM Tris base, 20mM acetic acid, 1 mM EDTA, pH 8.0
ddH2O	100mL
Agarose powder	1g (1g/1L of gel)
10,000X YeaRed Nucleic Acid Gel stain	10μL(10μL/block of gel)
6X Loading buffer	20μL(10μL/trial)	Contains bisphenol blue
Cas12a PCR System	250 (50μL/trial)	See “Target Protein DNA PCR” for specifics

   Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Agarose gel electrophoresis mold	1
Agarose gel electrophoresis mold comb	1	13 holes total
Pipette	1	Capacity 1μL-50μL
Gel electrophoresis machine	1	Gel block oriented horizontally; DNA runs parallel to the ground, distinct from SDS-PAGE setup
Measuring cylinder	1	Capacity ≥ 100mL
Conical flask	1	Capacity ≥ 100mL

 

Gel Block Preparation:  

1. Measure 1 L ddH₂O using the measuring cylinder and transfer to a conical flask.  
2. Add 1 bag of TAE buffer (~18.4 g) to the ddH₂O in step 1.
3. Add 1g of agarose powder to the ddH₂O in step 1.   
4. Mix the solution thoroughly by shaking the flask until all added powders are dissolved.  
5. Pour the solution into the gel electrophoresis mold.  
6. Place the comb into the gel electrophoresis mold and fix it in place in the designated slots. Wait for the gel to solidify.
7. System Preparation:  
8. Add 10 μL of 6X loading buffer to the PCR system from the previous procedure.  
9. Mix evenly.  
10. Repeat steps 7-8 for all PCR systems across all trials.  
11. Electrophoresis:  
12. Place the gel block into the gel electrophoresis machine. Ensure that the side of the gel block with holes is placed opposite to the cathode.
13. Add the 1X TAE buffer to the electrophoresis setu[
14. Using the pipette, add 20μL of 2k marker to the topmost hole. 
15. Add 50μL of the cas12a PCR system into the subsequent holes.  
16. Repeat step 13 for both PCR systems.  
17. Repeat steps 13-14 for all trials.  
18. Set the gel electrophoresis machine to 150 V and allow it to run for 20-30min. Stop once the bisphenol blue in the loading buffer has reached near the end of the gel block.  

 

1.4 DNA Recovery From Gel of Cas12a

 

Material statistics:

Material Name	Quantity	Notes
Anhydrous ethanol	1500μL(500μL/trial)
B2 buffer	1800μL(600μL/trial)
Wash solution	4500μL(1500μL/trial)

  Equipment statistics:

Equipment Name	Quantity	Notes
Water Bath	1
Gel transilluminator	1
Scalpel	1
Pipette	≥1	Capacity between 10μL-100μL
Pipette	≥1	Capacity between 100μL-1000μL
Centrifuge	1

 

Preparation:  

1. Check whether there is sediment in Buffer B2 
2. Add 500μL of anhydrous ethanol to the wash solution 
3. Adjust the water bath pot to 50ºC  
4. Gel Liquification 
5. Place the block of gel under the gel transilluminator. [1] 
6. Cut out the gel block containing the target fragment from the agarose gel using the scalpel and weigh it. Record the mass 
7. Transfer the gel containing the target DNA to an EPtube. Dispose of the rest of the gel block.  
8. Add 600μL B2 buffer to the EP tube containing the target DNA to the agarose gel block and place the EP tube in a 50ºC water bath for 11 minutes. [2] 
9. Transfer the solution from step 7 into the binding column, centrifuge at 8000rpm for 30 sec, then discard the eluate. [3] 
10. DNA Extraction:  
11. Add 500μL wash solution, then centrifuge the EP-tube-binding-column complex at 9000rpm for 30 seconds. Discard the eluate. [4] 
12. Repeat step 9 once 
13. Centrifuge the empty binding column [5] at 9000rpm for 1 minute 
14. Place the binding column into a clean 1.5mL EP tube and add 30μl of elution buffer to the adsorption film in the center of the binding column.  
15. Allow the elution buffer to sit at room temperature for 1 minute 
16. Centrifuge the binding-column-test-tube complex at 9000rpm for 1 minute and preserve the DNA solution in the tube. [6] 

 Note：

[1] This shows where the target DNA is in the gel. The DNA fluoresces due to the nucleic acid gel stain added in the previous procedure.  

[2] This liquifies the gel.  

[3] The centrifugal force pushes the solution through the adsorption film in the binding columns, leaving the DNA attached to the film.  

[4] This washes the film and removes unwanted materials from the adsorption film.  

[5] The binding columns still contain the BD-tau/T-tau DNA.  

[6] This removes the DNA from the binding column and suspends it in the elution buffer.  

 

1.5 Double Digestion of pET28a and cas12a

 

Material statistics:

Material Name	Quantity	Notes & Parameters
10X Buffer	15 μL (5 μL/trial)
Cas12a DNA	25 μL (25 μL/trial)	Recovered from gel in previous procedure
pET-28a	25 μL (25 μL/trial)	Recovered from gel in previous procedure
Hind III	3 μL(1μL/trial)	Hind III is an endonuclease that recognizes the following sequence: 5' A^AGCTT 3' [1]
Nhe I	3 μL(1μL/trial)	Nhe I is an endonuclease that recognizes the following sequence: 5' G^CTAGC 3'

 

  Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Micropipettes (P10, P20, P200)	1 set
Sterile pipette tips	1 box
Microtubes	3	1 per reaction + extra, capacity 200μL
Test tube rack	1
Microcentrifuge	1
Vortex mixer	1
Water bath	1	Maintain 37°C
Styrofoam box with ice inside	1	Enzyme/DNA storage
Pipette	1	Capacity between 1μL-20μL

 

System Preparation:  

1. Add 15 μL ddH₂O, 10μL of the target type of DNA for double digestion, 5μL of the 10X buffer, 1 μL Hind III and 1 μL Nhe I to a PCR tube.  
2. Repeat step 1 for both remaining types of DNA subject to double digestion (Cas12a /pET-28a).  
3. Place all samples into microcentrifuge for 30sec.  
4. Incubation:  
5. Place all samples into test tube rack.  
6. Keep these solutions in the water bath and allow to incubate for 2h at 37ºC.  

 

1.6 Ligation  of pET28a and cas12a

Material statistics:

Material Name	Quantity per Trial	Notes & Parameters
10X buffer	2μL
T4 ligase:	1μL
Cleaved cas12a DNA sequences	3 μL
Cleaved pET-28a	1μL
ddH2O	13μL
PCR tubes	3

Ligated Solution Preparation:  

1. Set the water bath to 50ºC and wait for its temperature to reach 50ºC.  
2. Place the PCR tubes containing the double digestion solution in the water bath set to 50ºC for 15min. [1] 
3. System Preparation: 
4. Add 13μL ddH₂O, 3μL of the target DNA sequence, 2μL of the 10X buffer and 1μL of pET-28a to an EP tube.[2]  
5. Repeat step 1 for the remaining sequences subject to ligation (Cas12a).  
6. Place all PCR tubes in microcentrifuge for 30sec 
7. Incubate in PCR machine for 1 hour under 16ºC 

Note：

[1] “^” represents the site of cleavage.  

[2] This denatures Nhe I and Hind III.  

 

 

1.7 Heat shocking 

 

Material statistics:

Material Name	Quantity	Notes & Parameters
BL21 culture
DH5a culture
PET-28a-cas12a	10μL (5μL/trial)
LB broth	≥3600μL (900μL/sample)	The broth should be sterile and should not contain kanamycin.
CaCl2

 

  Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Cooler	1	Generates temperatures of -80ºC or lower
Water bath	1	Temperature range includes 42ºC
Petri dishes	≥6	Prepared during LB preparation step; 0.1% kanamycin

 

Storage:  

1. Store the competent E.coli in a fridge set to -80ºC.  
2. Sample Preparation:  
3. Add 5μL of pET-28a solution carrying the cas12a gene to the BL21/DH5α e.coli samples and gently mix.  
4. Repeat step 2 three additional times to generate a total of four samples, representing all possible combinations of one E. coli strain with one gene (BL21 + BD-tau, BL21 + T-tau, DH5α + BD-tau, DH5α + T-tau). [1] 
5. Heat Shocking & Culturing:  
6. Place the microtubes containing the e.coli samples into a 42ºC water bath for 45sec. [2] Quickly transfer the samples to an ice bath to cool.  
7. Add 900 μL of kanamycin-free LB broth each microtube. Mix evenly by repeatedly drawing in and dispensing the LB using the pipette.  
8. Once the e.coli-LB solutions are evenly mixed, centrifuge at 8000rpm for 5min. 
9. Remove 900 μL of supernatant from all microtubes.
10. Use the samples to inoculate petri dishes. Allow the E.coli colonies to develop for 12-16h at 37ºC.
11. Preparation for PCR:
12. Pick random colonies in the culture & split each sampled colony into 2 equal portions; one will be used for PCR later the other inoculate LB broth [3] which contains 0.1% kanamycin from the LB preparation step [4].

 Note:

[1] 2 strains were cultured to find a (relatively) optimal strain for expressing the target proteins.  

[2] This sudden change in heat increases membrane permeability & makes the e.coli cells competent.  

[3] The petri dishes will be used to verify transfer success later, and the e.coli in the broth will be used for protein expression.  

[4] The pET-28a plasmid which will be used as a vector for the target genes later in the procedure encodes a gene which offers kanamycin resistance. This helps verify transformation success, as only e.coli which have received the kanamycin-resistance gene encoded on the pET-28a plasmid will survive the kanamycin.  

 

 

2.0  PCR For Target Genes Inside E.coli 

 

Material statistics:

Material Name	Quantity per Trial	Notes & Parameters
Buffer 2X	50 μL (5μL /sample)
F-primer	2 μL (0.2μL/sample)
R-primer	2 μL (0.2μL/sample)
ddH2O	46 μL (4.6μL/sample)
BL21 e.coli carrying cas12a DNA	N/A [1]	Engineered for superior protein-expressing capabilities
DH5a e.coli carrying cas12a DNA	N/A	Engineered for superior transformation efficiency

   Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Cooler	1	Generates
Microtubes	16	4 for each combination of DNA and e.coli strain

 

Preparing the System:  

1. Add 4.6T μLddH₂O, 5.0T μLbuffer, 0.2T μL F primer and 0.2T μL R primer to a test tube, where T is the number of desired trials. In this experiment, T=10.
2. Separate the system equally into T 10 μL portions.  
3. Gently scrape e.coli colonies off the petri dishes used to cultivate e.coli after the heat-shocking procedure using a pipette[1] .
4. Transfer some colonies to the microtubes containing the PCR solution. Swap the pipette tip between each culture. [2] 
5. Repeat steps 4-5 for all cultures.  
6. PCR:  
7. Preheat to 95ºC for 5min 
8. Heat to 95ºC for 15sec 
9. Cool to 65ºC for 15sec 
10. Heat to 72ºC for 10sec  
11. Repeat steps 8-10 30 times 
12. Hold temperature at 72ºC for 10min 

 Note:

[1] The colonies were extracted by scraping the petri dishes with a pipette tip, as opposed to being added as a solution, thus making their volume negligible.  

[2] This prevents cross-contamination.

 

 

2.1 Monitoring E.coli Growth Curve 

Material statistics:

Material Name	Quantity	Notes & Parameters
LB	8mL (2mL/test tube)	Contains 50ng/mL Kan+ (kanamycin)

   Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Shaker	1	The shaker is sealed and conditioned
Timer	1	Optional, a clock may be used instead
Spectrophotometer	1	500μL cuvette recommended
Test tube	4	Capacity 15mL (2 samples/test tube)
Pipette	≥1	Capacity between 100μL-1000μL

 

 

Sample Preparation:  

1. Mingle 2mL LB (K+ with 50ng/ml concentration) with 50 μL pET18a-cas12a E. coli cultured in the heat shocking step. Mix evenly
2. Place all tubes into 37˚C shaker. 
3. Observation:  
4. Return to record data for OD600 at different points for both proteins. (0.5, 1, 2, 4, 6, 8, 12 and 24h) [1] 
5. OD 600 Protocol:  
6. Remove the cuvette from the spectrophotometer & clean its exterior if needed.  
7. Add 500μL of sterile LB to the spectrophotometer cuvette.  
8. Place the cuvette back into the spectrophotometer 
9. Measure the OD 600 value of the blank. [2] 
10. Remove & empty the cuvette.   
11. Transfer the culture to the cuvette and measure the absorbance. 
12. Place the cuvette back into the spectrophotometer. 
13. Measure the OD-600 of the sample.  

• Repeat steps v-viii for all samples.  
• Note:

[1] OD600 is an approach which measures the light absorbance of bacterial or yeast cultures at 600nm wavelength using a spectrophotometer. As the curve requires infrequent, periodic measurements, this procedure can take place alongside other procedures.  

[2] This pares the spectrophotometer. 

 

2.2 Protein Extraction & Purification

 Material statistics:

Material Name	Quantity	Notes & Parameters
Non-denaturing lysis buffer	5mL
Wash solution	3mL (500μL/Wash)
Ice

  Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Centrifuge	1	Can generate 1000rpm--10000rpm
Sonicator	1
Beaker	1

 

Sonification Lysis Preparation:  

1. Centrifuge the tubes containing the E. coli (pET28a-cas12a) culture at 6000rpm and 4˚C for 15 minutes to concentrate the E. coli into a pellet.  
2. Extract & discard the supernatant using a pipette.  
3. Add 20mL non-denaturing lysis buffer to the pellet. [1] 
4. Vortex the solution to mix thoroughly.  
5. Sonification Lysis:  
6. Uncap the test tube containing the E.coli culture and place it into a beaker containing ice. [2] 
7. Place the beaker on the sonicator’s platform with the test tube’s opening positioned directly under the needle and raise the platform until the needle is touching the solution. Ensure that the needle is not touching the test tube walls. [3] 
8. Sonicate at 300 W with 2 s on/2 s off cycles for 30 min.  
9. Remove the test tube from the sonicator once sonication is complete.  
10. Repeat steps 5-8 for T-tau.
11. Extracting Target Proteins:  
12. Centrifuge for 30 minutes at 10000 rpm at 4˚C.  
13. Extract the supernatant, transfer to an 15 mL test tube and place it on ice.
14. Extract 20 μL of the supernatant for SDS-PAGE later.  
15. Add 1 mL his-tag purification resin to an EP tube.
16. Centrifuge at 1000rpm at 4˚C for 10 seconds. Discard the supernatant. [4] 
17. Add 500 μL non-denaturing lysis buffer to the EP tube and centrifuge at 1000 rpm at 4˚C for 10 seconds. [5] 
18. Repeat step 15 2 more times.  
19. Add his-tag purification resin to test tubes containing BD-tau. Mix evenly.
20. Incubate for 60min at 4ºC on a shaker. 
21. Add 10mL of BD-tau supernatant and His-tag purification resin into large binding columns. [6] Keep the bottom capped.  
22. Allow protein solution containing BD-tau bound to his-tag purification resin to pass through the binding column. The resin beads should remain above the filter.
23. Add 500μL wash solution to the binding column and allow it to pass through the binding column film.  
24. Collect 20μL of the eluate for sampling later. Store the rest.  
25. Repeat steps 20-21 2 more times.  
26. Add 500μL elution buffer to the binding column and allow it to pass through the binding column film. 
27. Collect 20μL of the eluate for sampling later. Store the rest.  
28. Repeat step 23-24 3 more times.  
29. Collect the final eluate.  

Repeat steps 10-26 for T-tau
 Note:

[1] This suspends the e.coli in preparation for lysis 

[2] Sonication generates heat. The ice cools the sample to prevent heat-induced protein denaturation.  

[3] The needle and test tube would damage each other if in contact for extended periods during operation.  

[4] This leaves only the resin on the binding column film. The eluate is the storage solution for the resin.  

[5] The non-denaturing lysis buffer is used to wash the resin

[6] This binding column contains the his-tag purification resin beads and is larger than the binding columns used in the EP tubes.  

 

2.3 SDS-PAGE

 Material statistics:

Material Name	Quantity	Notes & Parameters
ddH2O
30% Acr-Bis
Gel buffer A	3.75 mL
Gel buffer B	2 mL
Mixing cups

 Equipment statistics:

Equipment Name	Quantity	Notes & Parameters

 

Sample Preparation:  

1. Add 10 μL protein sample and 10 μL 2X loading buffer to an EP tube.  
2. Heat the microtube to 95˚C for 5 minutes.
3. Casting SDS-PAGE Gel: 
4. Fix the gel casting cassette into the casting frames.  
5. Prepare the 15% resolving gel by adding 3.6ml ddH₂O, 7.5 mL 30% Acr-Bis, 3.75 mL Gel Buffer A, 0.15 mL 10% APS and 0.009 mL TEMED to a mixing cup.  
6. Pipette the resolving gel into the gel casting cassette.  
7. Add anhydrous ethanol to the gel casting cassette until the ethanol level reaches the height of the shorter plate 
8. Wait for 10-15min.  
9. Decant the ethanol.  
10. Prepare the 5% stacking gel by adding 1.33mL ddH₂O, 0.67 mL 30% Acr-Bis, 2 mL Gel Buffer B, 0.04 mL 10% APS and 0.004 mL TEMED to a mixing cup. 
11. Pipette the stacking gel into the gel casting cassette. 
12. Insert the comb into the gel casting cassette.  
13. Wait for 10-15min.  
14. Carefully remove the combs from the gel casting cassettes.  

Buffer Preparation:  

1. Prepare a conical flask with capacity 1 L or greater.  
2. Add 1 bag (approx. 18.4g) of tris-glycine SDS buffer into the conical flask.  
3. Measure 1 L of water using a measuring cylinder.  
4. Add the water to the conical flask. 
5. Mix evenly until tris-glycine SDS buffer is fully dissolved.  
6. SDS-PAGE Electrophoresis:  
7. Transfer the cassettes to an SDS-PAGE gel electrophoresis tub.  
8. Add to each gel block the following samples:  
9. Markers for the first 2 cells. For the remaining 9 cells, add 10 μL CL, FT, W1, W2, W3, E1, E2, E3 and E4 in order.
10. Add buffer to the space between the cassettes within the tank until the space is completely full.  
11. Place the cap onto the setup. Ensure that the anodes and cathodes are properly paired.  
12. Plug the cap’s wires into an electrophoresis power supply. Ensure that the anodes and cathodes are properly paired. 
13. Turn on the power supply. Set the voltage to 60 V for 35min. Then, set the voltage to 130 V for 40min.  
14. Gel Dyeing:  
15. Cut the stacking gel from the gel block using a scalpel and discard it.  
16. Place the resolving gel block into a dyeing tub and add 20 mL of Coomassie brilliant blue dye to the tub.  
17. Place the dyeing tub onto a shaker and allow to dye overnight.  

Place the dyed gel block into an SDS-PAGE gel reader and document the results.

3.1 Preparation of crRNA

 

Material statistics:

Material Name	Quantity	Notes & Parameters
crRNA-T7-promoter-F	300μL	10 pM
crRNA-T7-R	300μL	10 pM
Vitro Transcription Kit	1	/
RNA Clean-up Kit	1	/
RNase-free Water	50ml	/

 Equipment statistics:

Equipment Name	Quantity	Notes & Parameters
Spectrophotometer	1	/
PCR	1	/
RNase-free Laminar Flow Cabinet	1	/

Preparation Procedure:

1. According to the design, primers crRNA-T7-promoter-F and crRNA-T7-R, used for preparing the template DNA for crRNA synthesis, were synthesized by the company. The primers were dissolved in RNase-free water, and their concentrations were measured using a NanoDrop spectrophotometer and adjusted to 10 µM.
2. 9 µL of crRNA-T7-promoter-F, 9 µL of crRNA-T7-R, and 2 µL of 10x Taq DNA Polymerase PCR Buffer were aliquoted into a PCR tube. The mixture was vortexed and briefly centrifuged to collect the liquid at the bottom of the tube.
3. The tube was placed in a PCR machine. Step 1: Denaturation at 94 °C for 5 minutes. Step 2: 74 cycles were run with the temperature decreasing by 1 °C every 30 seconds, stopping at 20 °C. This yielded 20 µL of crRNA transcription template DNA.

After obtaining the crRNA transcription template DNA, the HiScribe T7 Quick High-Yield RNA Synthesis Kit was used to prepare the crRNA. The specific steps are as follows:

1. 20 µL of crRNA transcription template DNA, 1 µL of RNase Inhibitor, 5 µL of RNA Polymerase Mix, and 25 µL of dNTP mixture were aliquoted into a PCR tube. The mixture was vortexed and briefly centrifuged.
2. The tube was placed in a PCR machine and incubated at 37 °C for 15 hours.
3. 1 µL of DNase was added, and incubation continued at 37 °C for 1 hour. After the reaction, 50 µL of pre-purified crRNA was obtained.

After transcription, the crRNA was purified using the RNA Clean & Concentrator TM-5 kit. The specific steps are as follows:

1. The entire 50 µL of pre-purified crRNA was transferred to a 1.5 mL nuclease-free microcentrifuge tube. 100 µL of RNA Binding Buffer was added and mixed thoroughly, followed by the addition of 150 µL of 100% ethanol and thorough mixing.
2. The mixture was transferred to a silica spin column and centrifuged at 10,000 × g for 1 minute. The flow-through was discarded.
3. 400 µL of RNA Prep Buffer was added to the column, followed by centrifugation at 10,000 × g for 1 minute. The flow-through was discarded.
4. 700 µL of Wash Buffer was added to the column, followed by centrifugation at 10,000 × g for 1 minute. The flow-through was discarded. This washing step was repeated once.
5. The empty column was centrifuged at 10,000 × g for 2 minutes to remove residual ethanol. The column was then transferred to a new 1.5 mL nuclease-free microcentrifuge tube.
6. 50 µL of Nuclease-free Water was added directly to the silica membrane. After incubating for 2 minutes, the column was centrifuged at 10,000 × g for 2 minutes. The column was discarded, and the eluate (containing the purified crRNA) was collected, yielding 50 µL of purified crRNA.
7. The concentration of the purified crRNA was measured using a NanoDrop spectrophotometer and adjusted to 10 µM. After adjustment, the crRNA was aliquoted into PCR tubes, 20 µL per tube. The aliquots were stored at -80°C for future use.

(Note: Throughout the crRNA preparation experiment, nuclease-free consumables were used, and all procedures were performed in an RNase-free environment.)

4.1 Preparation of Aptamer-dsDNA

Material statistics:

Material Name	Quantity	Notes & Parameters
Tau aptamer-F	200μL	10μmol
dsDNA-F	200μL	10μmol
dsDNA-R	200μL	10μmol
Streptavidin magnetic beads	1ml	1ml
ComDNA	500μL	10μmol
PBST	500ml

 Equipment statistics:

Equipment Name	Quantity
NanoDrop	1
PCR	1
RNase-free Laminar Flow Cabinet	1
Magnetic Rack	3

Preparation Procedure:

1. The corresponding customized aptamer (Tau aptamer-F) and the forward/reverse primers (dsDNA-F and dsDNA-R) for generating the Cas12a-activating dsDNA were synthesized by the company and adjusted to a concentration of 10 μmol.
2. The three components were mixed in a 1:1:1 ratio in a solution containing 1× Taq Buffer and thoroughly mixed.
3. The mixture was incubated at 95 °C for 5 minutes, followed by gradual cooling to room temperature at a rate of 2 °C per minute.
4. The resulting aptamer-dsDNA complex was quantified using a NanoDrop spectrophotometer, diluted to a final concentration of 5 μM, and stored at -20°C for future use.

4.2 Beads-ComDNA/Aptamer-dsDNA Complex

Material statistics:

Material Name	Quantity	Notes & Parameters
Tau aptamer-F	200μL	10μmol
dsDNA-F	200μL	10μmol
dsDNA-R	200μL	10μmol
Streptavidin magnetic beads	1ml	1ml
ComDNA	500μL	10μmol
PBST	500ml

 Equipment statistics:

Equipment Name	Quantity
NanoDrop	1
PCR	1
RNase-free Laminar Flow Cabinet	1
Magnetic Rack	3

Preparation Procedure:

1. Take out the streptavidin magnetic beads stored at 4 °C and vortex thoroughly to ensure a homogeneous suspension.
2. Transfer 20 μL of the bead suspension to a nuclease-free 1.5 mL microcentrifuge tube. Place the tube on a magnetic rack for 1 minute to separate the beads from the storage solution. Carefully aspirate and discard the storage solution.
3. Add 200 μL of 1× PBST buffer, vortex to mix, then place the tube on the magnetic rack for 1 minute. Discard the supernatant. Repeat this washing step three times to thoroughly remove the NaN₃ preservative from the streptavidin magnetic bead storage solution.
4. After washing, add 20 μL of the pre-quantified, 15 μM biotin-modified ComDNA to the tube. Then add 1× PBST buffer to bring the total volume to 200 μL.
5. Vortex the mixture and then incubate on a rotator for 30 minutes to allow thorough binding between the ComDNA and the streptavidin on the beads.
6. After incubation, place the tube on the magnetic rack for magnetic separation. Aspirate and discard the supernatant to remove excess ComDNA that did not bind to the beads.
7. Add 200 μL of 1× PBST buffer, vortex to mix, place on the magnetic rack for 1 minute, and discard the supernatant. Repeat this washing step twice.
8. After washing, add 20 μL of the pre-prepared 5 μM aptamer-dsDNA complex to the tube. Add 1× PBST buffer to bring the total volume to 200 μL.
9. Vortex the mixture and then incubate on a rotator for 30 minutes to allow hybridization between the aptamer-dsDNA and the ComDNA on the beads.
10. After incubation, place the tube on the magnetic rack for magnetic separation. Aspirate and discard the supernatant to remove excess aptamer-dsDNA that did not bind.
11. Add 200 μL of 1× PBST buffer, vortex to mix, place on the magnetic rack for 1 minute, and discard the supernatant. Repeat this washing step three times.
12. After the final wash, add 900 μL of 1× PBST buffer to resuspend the "Beads-ComDNA/Aptamer-dsDNA" complex. Aliquot 18 μL of the suspension into individual PCR tubes for subsequent detection assays.

5.1 Biosensing platform optimization

Assessment of the Aptamer's Effect on Cas12a Activation by dsDNA

Goal: It is first necessary to verify whether the dsDNA can still be cleaved by Cas12a after binding to the aptamer.

Material statistics:

Material Name	Quantity	Notes & Parameters
Tau aptamer-F	200μL	10μmol
dsDNA-F	200μL	10μmol
dsDNA-R	200μL	10μmol
Streptavidin magnetic beads	1ml	1ml
ComDNA	500μL	10μmol
PBST	500ml

 Equipment statistics:

Equipment Name	Quantity
NanoDrop	1
PCR	1
RNase-free Laminar Flow Cabinet	1
Magnetic Rack	3

Preparation Procedure:

1. Prepare Tau aptamer-dsDNA solutions at different concentrations (5, 10, 15, 20, 25, 30 μmol). Use a dsDNA solution (30 μmol) as a positive control.
2. Add the three components in a 1:1:1 ratio to a solution containing 1× Taq Buffer and mix thoroughly.
3. Incubate the mixture at 95 °C for 5 minutes, then gradually cool to room temperature at a rate of 2 °C per minute.
4. Quantify the resulting aptamer-dsDNA using a NanoDrop spectrophotometer and store at –20 °C for future use.
5. Add the prepared samples to the Cas12a reaction system (e.g., a 2.5× Cas12a reporter assay system) and measure the time-dependent fluorescence intensity curves to confirm that the fluorescence intensity is positively correlated with the concentration of Tau/BD-tau aptamer-dsDNA.
   1. Screening of ComDNA for the Highest Signal-to-Noise Ratio

Goal: It is first necessary to verify whether the dsDNA can still be cleaved by Cas12a after binding to the aptamer.

Material statistics:

Material Name	Quantity	Notes & Parameters
Tau aptamer-F	200μL	10μmol
dsDNA-F	200μL	10μmol
dsDNA-R	200μL	10μmol
Streptavidin magnetic beads	1ml	1ml
ComDNA	500μL	10μmol
PBST	500ml
qPCR-F	10pM
qPCR-R	10pM
2XSYBR qPCR Mix	5ml
Tau	/	5mg/ml
BD-tau	/	5mg/ml

 Equipment statistics:

Equipment Name	Quantity
NanoDrop	1
PCR	1
RNase-free Laminar Flow Cabinet	1
Magnetic Rack	3
qPCR	1

Procedure:

1. Prepare five distinct ComDNA-Tau aptamer-dsDNA complexes according to the steps described in Section 4.2.
2. Add a 10 μmol excess of Tau protein/BD-tau and incubate at 4 °C for 60 minutes.
3. Searate the supernatant using a magnetic rack and store it at -80 °C.
4. Quantify the amount of released BD-tau/Tau aptamer-dsDNA complex using quantitative real-time PCR (qPCR). Use a control sample where an equivalent volume of water is added instead of Tau protein. Calculate the signal-to-noise ratio, using actin as the internal reference gene, to identify the ComDNA sequence yielding the highest signal-to-noise ratio.

qPCR Reaction Setup:

Component	Volume
2XSYBR qPCR Mix	12.5
Template	7
qPCR-F	7
qPCR-R	7
R-free H2O	16.5

qPCR ProgramqPCR Program:

Temperature	Time	Cycle
95°C	2:00
95°C	1:00	30
60°C	0:30
72°C	0:30
95°C	0:15
60°C	1:00
95°C	0:15
60°C	1:00

1. After adjusting the qPCR program, data were collected.
   1. Determination of the Concentrations of ComDNA and the Tau/BD-tau Aptamer-dsDNA Complex

Goal：The screening of aptamer-switched ComDNA requires the quantification of both the ComDNA and the Tau/BD-tau aptamer-dsDNA complex.

Material statistics:

Material Name	Quantity	Notes & Parameters
Tau aptamer-F	200μL	10μmol
dsDNA-F	200μL	10μmol
dsDNA-R	200μL	10μmol
Streptavidin magnetic beads	1ml	1ml
ComDNA	500μL	10μmol
PBST	500ml
qPCR-F	10pM
qPCR-R	10pM
2XSYBR qPCR Mix	5ml
Tau	/	5mg/ml
BD-tau	/	5mg/ml

 Equipment statistics:

Equipment Name	Quantity
NanoDrop	1
PCR	1
RNase-free Laminar Flow Cabinet	1
Magnetic Rack	3
qPCR	1

Procedure:

1. The "beads-biotinylated ComDNA-Tau aptamer-dsDNA" complex was constructed by adding different concentrations (1, 2, 5, 10, 20 μM) of ComDNA to the BD-tau/Tau aptamer-dsDNA, according to the protocol in Section 4.2.
2. Add a 10 μmol excess of Tau protein/BD-tau and incubate at 4 °C for 60 minutes.
3. Searate the supernatant using a magnetic rack and store it at -80 °C.
4. Quantify the amount of released BD-tau/Tau aptamer-dsDNA complex using quantitative real-time PCR (qPCR). Use a control sample where an equivalent volume of water is added instead of BD-tau/Tau protein. Calculate the signal-to-noise ratio, using actin as the internal reference gene.

qPCR Reaction Setup:

Component	Volume
2XSYBR qPCR Mix	12.5
Template	7
qPCR-F	7
qPCR-R	7
R-free H2O	16.5

qPCR ProgramqPCR Program:

Temperature	Time	Cycle
95°C	2:00
95°C	1:00	30
60°C	0:30
72°C	0:30
95°C	0:15
60°C	1:00
95°C	0:15
60°C	1:00

1. After adjusting the qPCR program, data were collected.

5.3. Determination of the Incubation Time for BD-tau/Tau with the ComDNA-Aptamer-dsDNA Complex

Goal: To optimize the incubation time of BD-tau/Tau protein with the sensor to ensure experimental success.

Procedure:

1. Using the ComDNA sequence and concentration screened in step 5.2, prepare the sensor according to the protocol in section 4.2.
2. Add 10 μM BD-tau/Tau and incubate for different time periods (10, 20, 30, 40, 50, 60 min) to assess the release of the BD-tau/Tau aptamer-dsDNA complex.
3. Perform quantitative analysis using qPCR. The PCR protocol is as follows.

qPCR Reaction Setup:

Component	Volume
2XSYBR qPCR Mix	12.5
Template	7
qPCR-F	7
qPCR-R	7
R-free H2O	16.5

qPCR ProgramqPCR Program:

Temperature	Time	Cycle
95°C	2:00
95°C	1:00	30
60°C	0:30
72°C	0:30
95°C	0:15
60°C	1:00
95°C	0:15
60°C	1:00

5.4 Quantitative Analysis of the Cas12a Reporting System

Goal：After determining the concentrations of ComDNA and the Tau aptamer-dsDNA complex as well as the incubation time, the complete sensor response and signal output procedure was performed to detect BD-tau/Tau protein.

Material statistics:

Material Name	Quantity	Notes & Parameters
Tau	/	5mg/ml
BD-tau	/	5mg/ml
Magnetic Beads-cDNA/Aptamer-dsDNA	2ml
Cutsmart buffer	2ml
Enzyme Inhibitor	5ml
crRNA	1ml	10 μM
Cas12a protein	1ml	10 μM
ss-DNA		10 μM

 Equipment statistics:

Equipment Name	Quantity
Multimode Microplate Reader	1
NanoDrop	1

Procedure:

1. Prepare a stock solution of standard concentration using BD-tau/Tau protein, and then perform a series of twofold dilutions to obtain protein samples of varying concentrations.
2. Add 2 μL of each gradient concentration sample to 18 μL of the "Magnetic Beads-cDNA/Aptamer-dsDNA" complex solution. Mix well and incubate at room temperature for 40 minutes.
3. After incubation, perform magnetic separation for 1 minute, and then transfer 10 μL of the supernatant to a new PCR tube.
4. Take 10 μL each of the post-reaction supernatant from the "Magnetic Beads-cDNA/Aptamer-dsDNA" complex solution (or standard concentrations of Cas12a-activating dsDNA).
5. Add 2 μL of each sample to the bottom of the wells in a microplate.
6. In a 200 μL PCR tube, prepare the detection mixture by adding the following:

Component	Volume
10x CutSmart buffer	10 μL
RNase inhibitor	1 μL
10 μM crRNA	0.5 μL
10 μM Cas12a protein	0.5 μL
10 μM ssDNA reporter probe	5 μL
RNA-free H20	73 μL

1. Vortex the mixture thoroughly to ensure homogeneity. Use a mini centrifuge to spin down any liquid on the tube walls.
2. Aliquot 9 μL of the detection reaction mixture into each pre-loaded well of the microplate. Dispense the liquid onto the left and right sidewalls of the wells.
3. Use a microplate centrifuge to spin the plate, ensuring the reaction mixture collects at the bottom of the wells.
4. Turn on the multimode microplate reader. Set the instrument to read every 1.5 minutes for a total duration of 60 minutes. Use an excitation wavelength of 520 nm and an emission wavelength of 480 nm.

5.5 Evaluation of Sensor Specificity and Effectiveness

Goal: For comparison, Bovine Serum Albumin (BSA), Human Serum Albumin (HSA), Immunoglobulin E (IgE), and Immunoglobulin G (IgG) can be selected for specificity detection. The fluorescence intensity is to be analyzed when the concentration of these analogs is 10 μmol (consistent with the Tau protein concentration).

Procedure:

1. Take 10 μmol solutions of BSA, HSA, IgE, and IgG.
2. Add 2 μL of each protein solution to 18 μL of the "Magnetic Beads-cDNA/Aptamer-dsDNA" complex solution. Mix well and incubate at room temperature for 40 minutes.
3. After incubation, perform magnetic separation for 1 minute, and then transfer 10 μL of the supernatant to a new PCR tube.
4. Take 10 μL of the post-reaction supernatant from each "Magnetic Beads-cDNA/Aptamer-dsDNA" complex solution mixture.
5. Add 2 μL of each supernatant to the bottom of individual wells in a microplate.
6. In a 200 μL PCR tube, prepare the detection mixture by adding the following:

Component	Volume
10x CutSmart buffer	10 μL
RNase inhibitor	1 μL
10 μM crRNA	0.5 μL
10 μM Cas12a protein	0.5 μL
10 μM ssDNA reporter probe	5 μL
RNA-free H20	73 μL

1. Vortex the mixture thoroughly and use a mini centrifuge to spin down the liquid collected on the tube walls.
2. Aliquot 9 μL of the detection reaction mixture into each pre-loaded well of the microplate. Dispense the liquid onto the left and right sidewalls of the wells.
3. Use a microplate centrifuge to spin the plate, ensuring the reaction mixture is centrifuged to the bottom of the wells.
4. Turn on the multimode microplate reader. Set the instrument to read every 1.5 minutes for a total duration of 60 minutes, using an excitation wavelength of 520 nm and an emission wavelength of 480 nm.

For cycle 3-5 the primary experiment steps are identical with those of cycle 1-2，so we do not duplicate the detailed steps here.

Attachments 1:

(1) ELISA Random Clinical Trial Generator

#include <iostream>

#include <iomanip>

#include <random>

#include <cstdlib>

#include <ctime>

#include <cmath>

int main(int argc, const char * argv[]) {

int varimax, varimin, solutionvol=200;

int trialnum;

std::cout << "Insert the number of trials needed:" << std::endl;

std::cin >> trialnum;

std::cout << "Insert the maximum and minimum concentrations for any given trial starting with the smaller parameter:" << std::endl;

std::cin >> varimin >> varimax;

double trials [trialnum+1];

srand(time(0));

for (int i=1;i<=trialnum;++i){

double random = varimin + rand()%(varimax-varimin);

trials [i] = random;

int conplace=0;

int concentrations [trialnum+1];

for (int constor = trials [i]/5;constor>0;constor/=10){

++conplace;

}

concentrations[i] = pow(10,conplace);

double substrate = (trials[i]/5)*2*pow(10,2-conplace);

std::cout << trials [i] << " " << 5 * concentrations [i] << " " << substrate << " "<< solutionvol-substrate << std::endl;

//std::cout << conplace << std::endl;

//std::cout << 5*concentrations [i] << std::endl;

}

return 0;

}