Team:NYU Abu Dhabi/Experiments

EXPERIMENTS

What is DETECTR?
DNA endonuclease-targeted CRISPR trans reporter (DETECTR) is a novel technique combining DNA amplification by Recombinase Polymerase Amplification (RPA) at a constant temperature of 39 °C followed by detection with the CRISPR-Cas12a system. This technique is used to detect the presence of a certain target DNA.

Firstly, RPA amplifies the target DNA to yield a high number of copies. Next, the CRISPR guide RNA, which is complementary to the target DNA, binds to it, activating the Cas12a enzyme and causing it to begin cis cleavage (for the bound DNA) and the trans cleavage (for other DNA in the solution).

Within the solution, the FQ quencher, which releases fluorescence upon being cut by the Cas12a enzyme, sends a light signal when the target DNA is present. The higher the amplification using RPA, the stronger the fluorescence signal of the quencher.

Experimentation with DETECTR

Figure 1: Overview of Experimentation



In order to verify the functionality of the DETECTR (RPA+CRISPR) protocol and adjust it to the needs of the device, we conducted several tests in the lab regarding its sensitivity, specificity, and fluorescence emission. We also simulated tests on saliva sample by testing the technique directly on bacteria obtained from LB broth.

First, we began our experiments by ligating the gBlocks we selected on the pJET 1.2 backbone for all the genes, including pcaA (Tuberculosis), HBcAg (Hepatitis B), IS481 (Whooping Cough), and ypo2088 (Plague).

Next, we tested the sensitivity of Recombinase Polymerase Amplification (RPA) as compared to that of the currently-used method for diagnosis, Polymerase Chain Reaction (PCR). The results, which yielded a higher sensitivity for RPA compared to PCR, indicates that the protocol Volatect utilizes, DETECTR, is more sensitive than current diagnostic methods.

The fluorescence optimization aim was two-fold. First, we optimized SYBR green for use as an amplification verification tool. After conducting PCR tests on the bacteria from broth, for instance, we verified that the amplification was successful by checking for fluorescence when combined with SYBR green. Next, we tested an alternative to SYBR green with stronger fluorescence, FQ quencher. After CRISPR detects the targeted pathogens, it cuts the DNA, including the fluorescent probe (FQ quencher), which consequently releases fluorescence. Using CRISPR coupled with quencher, therefore, offers us more accurate detection with stronger fluorescent signal that can easily be detected by the sensors in the device.

The specificity of the DETECTR method and the multiplexing capacity of the system was also tested to verify the technique’s accuracy, which yielded positive results.

Finally, to simulate testing on saliva samples with the targeted pathogens, we conducted PCR and RPA tests followed by CRISPR and quencher directly on bacteria obtained from broth. The test results showed the RPA was more sensitive than PCR, and the fluorescence generated by the quencher after RPA was clearer and stronger.


Sample Preparation

PJET Ligation

Materials needed: Thermo Scientific CloneKet PCR Cloning Kit, nuclease-free water, pJET 1.2/blunt cloning vector

 

Methods:

  1. Prepare the Thermo Scientific CloneKet PCR Cloning Kit reagent mix in a PCR tube on ice.
  2. Add 10μL of 2X Reaction Buffer.
  3. Add 1μL of the resuspended gBlock gene fragment of interest.
  4. Add 1μL pJET 1.2/blunt cloning vector (50ng/μL).
  5. Add 7μL nuclease-free water.
  6. Add 1μL T4 DNA Ligase.
  7. Incubate at room temperature for 16 hours.
  8. Repeated for other gBlocks.

 

Transformation of E.coli via heat shock

Materials needed: Electrocompetent E. Coli (DH5⍺) cells, ligated pJET vectors, SOC (ThermoFisher), shaker Thermocycler (Eppendorf), centrifuge

 

Methods:

  1. Aliquot 200μL of electrocompetent E.coli (DH5⍺) into 4 eppendorf tubes.
  2. Add 10μL of ligated pJET vectors into each of 4 eppendorf tubes.
  3. Ice for 20 minutes, followed by a 60 second heat shock in an Eppendorf Thermocycler at 42°C.
  4. Return tubes to ice for 2 minutes.
  5. Add 800μL of S.O.C (ThermoFisher Scientific #15544034).
  6. Incubate on a shaker at 220 rpm and 37°C for one hour.
  7. Centrifuge for 1 minute at 13000 rpm and discard 700μL of the supernatant.
  8. Carefully resuspend the pellet and plate on LB+Ampicillin agar plate.
  9. Spread gently using a sterile inoculation loop.
  10. Incubate overnight (for 16-24 hours) at 37 °C.



Inoculation of Colonies After Transformation

Materials needed: Falcon tubes, LB+Amp broth, Inoculation loop 

 

Method:

  1. Add 10mL LB broth to 15mL culture tubes.
  2. Use a plastic inoculation loop to pick up a colony from each plate and swirl the colony in the broth to release it. Record the selected colonies for reference.

 

  1. Keep the tubes loosely capped and incubate on a shaker at 220 rpm at 37°C overnight (16-24 hours).

 

 Resuspension of Lyophilized PCR Primers from IDT

  1. Add volume of water indicated on order sheet (differed for each primer) to make 100 μM of each primer
  2. Pipetted up and down to mix



Miniprep of Inoculations (from the QIAGEN protocol)

Materials needed: Eppendorf tubes, P1 Buffer (Qiagen) , P2 Buffer (Qiagen), N3 Buffer (Qiagen), PE Buffer (Qiagen), EB Buffer (Qiagen), QIAprep spin column (Qiagen), centrifuge machine.  

 

Method:

  1. Centrifuge 5 ml of bacteria-broth solution from overnight culture at 7830 rpm (6800 x g) for 3 minutes at room temperature.
  2. Resuspend the pelleted bacterial cells in 250 μl of Buffer P1 and transfer to a microcentrifuge tube.
  3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 minutes.
  4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times.
  5. Centrifuge for 10 minutes at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
  6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 60 s and discard the flow-through.
  7. Add 0.5 ml of Buffer PB. Centrifuge for 60 seconds and discard the flow-through.
  8. Wash the QIAprep spin column by adding 0.75 ml Buffer PE. Centrifuge for 60 seconds and discard the flow-through.
  9. Centrifuge for 1 min to remove residual wash buffer.
  10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of the QIAprep spin column, let stand for 1 minute, and centrifuge for 1 minute.
  11. Nanodrop the miniprep sample using Thermo Scientific Nanodrop 2000C
  1. Blank a Nanodrop machine using 2μl of EB buffer for calibration. 
  2. Measure the nucleic acid concentration of the miniprep sample by loading 2μl of the sample on the cuvette. 
  3. Wipe the cuvette after each measurement. 



Serial Dilutions

Materials needed: EB Buffer (Qiagen), pipettes, pipette tips

 

Method:

  1. The serial dilutions of the miniprep samples were done as follows:
  1. Ypo2088 (Miniprep sample 3)

 

Serial Dilutions Number

Concentration (ng/µl)

Volume taken out of previous serial dilution (μl)

Volume of dH2O Added (μl)
 

Highest Concentration (x ng/µl)

77.5

  

1

60

19.35483871 (from Miniprep stock)

5.64516129

2

25

10.41666667

14.5833333

3

10

10

15

4

5

12.5

12.5

5

1

5

20

6

0.1

2.5

22.5

7

0.01

2.5

22.5

8

0.001

2.5

22.5



  1. HbcAg (Miniprep Sample 2)

 

Serial Dilutions Number

Concentration (ng/µl)

Volume taken out of previous serial dilution (μl)

Volume of dH2O Added (μl)
 

Highest Concentration (x ng/µl)

90

  

1

60

18  (from Miniprep stock)

9

2

25

10

14

3

10

10

15

4

5

12.5

12.5

5

1

5

20

6

0.1

2.5

22.5

7

0.01

2.5

22.5

8

0.001

2.5

22.5

 

  1. IS481 (Miniprep sample 2)



Serial Dilutions Number

Concentration (ng/µl)

Volume taken out of previous serial dilution (μl)

Volume of dH2O Added (μl)
 

Highest Concentration (x ng/µl)

129.7

  

1

60

11.56515035 (from Miniprep sample)

13.4348496

2

25

10.41666667

14.5833333

3

10

10

15

4

5

12.5

12.5

5

1

5

20

6

0.1

2.5

22.5

7

0.01

2.5

22.5

8

0.001

2.5

22.5

 

  1. pcaA (Miniprep Sample 1)

 

Serial Dilutions Number

Concentration (ng/µl)

Volume taken out of previous serial dilution (μl)

Volume of dH2O Added (μl)
 

Highest Concentration (x ng/µl)

111.9

  

1

60

13.40482574 (from Miniprep Sample)

11.595174

2

25

10.41666667

14.5833333

3

10

10

15

4

5

12.5

12.5

5

1

5

20

6

0.1

2.5

22.5

7

0.01

2.5

22.5

8

0.001

2.5

22.5



PCR

Optimized Bio Rad PCR Mix Protocol

Materials needed: BioRad Mastermix (BioRad: #1665009EDU), DNA sample, forward and reverse PCR primers for target gene, nuclease-free water

  1. Prepare 10 μM working stock of each primer by extracting 5 μl of 100 μM stock and adding 45 μl of distilled water to a total of 50 μl.
  2. Labelled each PCR tube with name of gBlock
  3. Add 20 μll PCR Master Mix (BioRad:  #1665009EDU) to each tube
  4. Add 1 μll of 10 μM of the forward primer to each appropriate tube. This is done for all the gBlocks..
  5. Add 1 μl of 10 μM of the reverse primers to each appropriate tube. This is done for all genes.
  6. Add 2μl of the gBlock template DNA.
  7. Add 16 μl of nuclease-free water to reach a total volume to 40 μl 
  8. Set the temperature of each part of the cycle according to the BioRad (35 cycles):
  1. Cycle 1: 94 °C for 2 min
  2. Cycle 2 (35 repeats): 94 °C for 1 min

60 °C for 1 min

72 °C for 2 min

  1. Cycle 3: 72 °C for 10 min
  2. Hold: 4°C overnight

 

Optimized NEB PCR Protocol (NEB Q5 High-Fidelity 2x Master Mix)

Materials needed: NEB Q5 High-Fidelity 2x Master Mix, primers, DNA template, nuclease-free water, PCR machine, pipette 

 

Method:

The following mix is prepared:

  1. 12.5 μl of NEB Q5 High-Fidelity 2X Master Mix
  2. 1.25 μl of forward primer (10 μM)
  3. 1.25 μl of reverse primer (10 μM)
  4. 2 μl of DNA template
  5. 8 μl of nuclease-free water

 

The following settings were inputted to the machine:

  1. Denaturation: 98 C for 30 sec
  2. Cycles (35): 
  1. 98 C for 10 seconds
  2. annealing temperature specific to the gene for 30 seconds
  3. 72 C for 15 seconds
  1. Final Extension: 72 C for 2 min
  2. Hold: 4 C 

 

Optimized NEB PCR Protocol (Taq 2x Master Mix)

  1. Add the following components in each pcr tube
    1. Taq 2X Master Mix: 8ul
  1. Forward Primer (10uM): 0.32ul
  2. Reverse Primer (10uM): 0.32ul
  3. Template DNA: 5ul
  4. Nuclease-free water: 2.36ul (up to 16ul)
  1. Set the temperature of each part of the cycle according to the BioRad (30 cycles):
  1. Cycle 1: 94 °C for 2 min
  2. Cycle 2 (35 repeats): 94 °C for 1 min

60 °C for 1 min (annealing temperature of primer)

72 °C for 2 min

Cycle 3: 72 °C for 10 min

  1. Hold: 4°C 



RPA 

Materials needed: TwistDx Basic Kit, nuclease-free water, RPA primers, Thermocycler

 

Method:

  1. Prepare the reaction mix in an eppendorf tube:
  2.  Add the following reagents to the reaction mix:
  1. Primer A (10µM) - 9.6µL
  2. Primer B (10µM) - 9.6µL
  3. Rehydration Buffer - 119µL
  4. dH2O - 32.8µL
  1. Pipette up and down to ensure proper mixing.
  2. Add the reaction mix in each tube to four freeze-dried reaction tube (provided by TwistDX Basic Kit).
  3. Add 16 μl of the reaction mix to 9 eppendorf tubes.
  4. Add 5µL of template from the serial dilutions (from the highest concentration to the lowest concentration) to the corresponding tubes. Leave one as a negative control.
  5. Add 1µL of 280mM magnesium acetate to each eppendorf tube and vortex to start the reaction.
  6. Incubate the reaction at 38 °C for 20 min using the Eppendorf Thermocycler.



CRISPR Cas12a non-specific cleavage

Optimized NEB CRISPR Protocol

Materials needed: NEBuffer 2.1 Reaction Buffer (10x), gRNA, EnGen Lba Cas12a (Cpf1), custom Fluorescence-Quencher(FQ) reporter (50μM), ready-made RPA reaction, incubator

 

Method:

Assemble the reaction at room temperature in the following order*:

  1. Add the following reagents in this order:
  • 2µl NEBuffer 2.1 Reaction Buffer (10x)
  • 0.5µl 5uM gRNA
  • 1.5µl 1 µM EnGen Lba Cas12a (Cpf1)
  1.   Pre-incubate the mixture for 10 minutes at 37⁰C
  2.   Add the RPA reactions
  3.   Incubate for 30 minutes at 37°C. Check the fluorescence every 10 minutes using E-gel Imager (ThermoFisher Scientific #4466612).
  4. Add 1ul of 0.5X FQ quencher to the CRISPR mix
  5. Add CRISPR reagents mix with quencher to RPA reaction mix
  6. Incubate 20 minutes at 37°C.
  7. Check under the blue light based E-gel imager.





Gel Electrophoresis

Preparation of 1x TAE buffer

Materials needed: Measuring cylinder, 50X TAE buffer, distilled water

Method:

  1. Add 20 ml of 50x TAE Buffer (BioRad #1660742EDU) into a 1000 ml measuring cylinder
  2. Dilute to 1000 ml using 980 ml of distilled water
  3. Mix to ensure homogeneity

 

Agarose Gel Preparation

Materials needed: agarose powder, 1x TAE buffer, scale, microwave, mittens, conical flask, GelRed, pipette, comb, cast

 

Method:

  1. Agarose powder was added to TAE buffer in a conical flask according to the following requirements:
    1. For 1% (PCR sample), 0.5 g of agarose powder was added to 50 ml of 1x TAE buffer
    2. For 3% (RPA sample), 1.5 g of agarose power was added to 50 ml of 1x TAE buffer 
  2. The solution was placed in a microwave, taken out and swirled during boiling. This was repeated until the solution became clear (after the third time boiling)
  3. 3 μl of Gel Red were added to the solution while swirling
  4. A well comb was added to the gel cast, the solution was poured into the gel cast and allowed to solidify for 20 minutes

 

 

REMOVED PARTS

---------------------------------------------

Loading of PCR Samples for Agarose Gel Electrophoresis to Confirm Proper Ligation

 Materials needed: eppendorf tubes, loading dye, ladder/ ruler, agarose, TAE Buffer, weighing boat, sensitive balance, microwave, graduated cylinders, beakers, pipettes, pipette tips. 

 

Method:

  1. Add 20 5 μl drops of loading dye onto paraffin paper.
  2. Add 10 μl of samples from each PCRed miniprep to each drop of loading dye.
  3. Load 5 μl of ladder (with dye) first, followed by 15 μl of samples (including the dye)
  4. Allow the gel to run for 20 minutes.
  5. View the gel
  1. Using the E-gel Imager (ThermoFisher Scientific #4466612) 
  2. Under UV light




RPA Sample loading for Gel Electrophoresis (3% Agarose)

  1. Add 5 μl drops of sample from each RPA reaction and 100 bp ladder onto a piece of parafilm.
  2. Add 1 μl of purple loading dye to each sample drop and mix by pipetting up and down.
  3. Load the ladder first, followed by 6 μl of each of the samples (including the negative control)
  4. Leave the gels to run for 20 minutes.
  5. View the results using:
  1. E-gel Imager
  2. UV light

 

Test SYBR Green on RPA Samples

Materials needed: 10000x SYBR green, RPA samples UV light machine, TE buffer

 

Method:

  1. Dilute SYBR Green 10,000X to 1,000X using TE buffer
  2. Add SYBR Green to yesterday's IS481 RPA samples (both 4µL and 10µL DNA).
  • Add 0.56µL of SYBR Green to RPA sample with 4µL of DNA
  • Add 0.80µL of SYBR Green to RPA sample with 10µL of DNA
  1. Test the fluorescence under UV light
  2. Dilute SYBR Green to 50X and 100X:
  • 50X SYBR Green: Add 5µL of 1000X SYBR Green to 95µL of TE buffer
  • 100X SYBR Green: Add 10µL of 1000X SYBR Green to 90µL of TE buffer

 

NEB PCR Protocol

Materials needed: NEB Q5 High-Fidelity 2x Master Mix, primers, DNA template, nuclease-free water, PCR machine, pipette 

 

Method:

The following mix is prepared:

  1. 12.5 μl of NEB Q5 High-Fidelity 2X Master Mix
  2. 1.25 μl of forward primer (10 μM)
  3. 1.25 μl of reverse primer (10 μM)
  4. 2 μl of DNA template
  5. 8 μl of nuclease-free water

 

The following settings were inputted to the machine:

  1. Denaturation: 98 C for 30 sec
  2. Cycles (35): 98 C for 10 sec, 50-72 C (annealing temp may differ) for 30 seconds, 72 C for 15 sec
  3. Final Extension: 72 C for 2 min
  4. Hold: 4 C overnight




NEB protocol for Taq 2x Master Mix

Materials needed: Taq 2X Master Mix, forward and reverse primer (10 μM), DNA template, nuclease-free water, PCR machine

 

Method:

Add the following components in each pcr tube

  1. Taq 2X Master Mix: 8 μl
  2. Forward Primer (10μM): 0.32μl
  3. Reverse Primer (10μM): 0.32μl
  4. Template DNA: 5μl
  5. Nuclease-free water: 2.36 μl (up to 16μl)

 

Thermocycling following BioLab protocol

  1. Initial Denaturation: 95°C 30 seconds
  2. 30 cycles
  • 95°C 20 seconds (15-30 seconds)
  • *60°C 30 seconds (15-60 seconds)

*Annealing temperature of the gene

  • 68°C 30 seconds
  1. Final extension: 68°C 5 minutes
  2. Hold: 4°C