iGEM 2023 — Team missouri-miners

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

# Date: 04/20/2023

Names: Danielle Dotson, Molly Ripper, Chambre Garcia, Matthew Gaston, Austin Thrasher

Start Time: 1:30 pm

Purpose: In Vitro transcription to produce LwCas13a-associated crRNA and miRNA-424-5p

## [Protocol:](https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0012652_TranscriptAid_T7_High_Yield_Transcription_UG.pdf)

(page 6)

### Set-Up

* Added 1125 µL of MilliQ water to 2023MissouriMinersT7CRRNA424 (crRNA). Results in a concentration of 4 µg/µL in the original DNA tube.
* Added 615 µL of MilliQ water to 2023MissouriMinersT7MIRNA424 (miRNA). Results in a concentration of 4 µg/µL in the original DNA tube.
* Thaw all frozen reaction components, mix, and centrifuge briefly to collect all drops.
* Keep TranscriptAid Enzyme Mix and nucleotides on ice.
* Keep the 5X TranscriptAid Reaction Buffer at room temperature.
* Combine equal volumes of the four NTP solutions in one tube for convenience and reduction of pipetting steps

1. Combine the following reaction components at room temperature in the order given:

   | Compoent | Amount |
   | --- | --- |
   | DEPC-treated water | to 20 µL → **5.5 µL** |
   | 5X TranscriptAid Reaction Buffer | **4 µL** |
   | ATP/CTP/GTP/UTP mix\* | **8 µL**b |
   | Template DNA | 2 µg → **0.5 µL** |
   | TranscriptAid Enzyme Mix | **2 µL** |
   | Total volume: | 20 µL |

   \* Equal volumes of the four provided NTP solutions combined in one tube.

    Tubes: (miRNA, trans, 4/20) and (crRNA, trans, 4/20)

## Results:

We should have a tube of miRNA 424-5p and a tube of crRNA for the Cas13 enzyme.

Notes: None

Stop Time: 7:00 pm

Next Time: Purification of RNA transcripts.

---

# Date: 04/20/2023

Names: Molly Ripper, Chambre Garcia, Matthew Gaston, Austin Thrasher

Start Time: 7:30 pm

Purpose: RNA Purification of transcription products from the last experiment

## [Protocol:](https://www.mn-net.com/media/pdf/b0/51/ee/Instruction-NucleoSpin-RNA.pdf)

1. Add RNase-free H2O to each of our 20 µL tubes of product from the in vitro transcription experiments up to 100 µL.
2. In two 1.5 ml centrifuge tubes, prepare a Buffer RA1/ethanol premix with a ratio of 1:1. 300 μL Buffer RA1 and 300 μL ethanol (96–100 %) were mixed for each 100 μL RNA sample.
3. Add 600μL of Buffer RA1/ethanol premix to 100µL of sample RNA and vortex to mix
4. A NucleoSpin® RNA Column was placed in a collection tube. Pipette the lysate up and down 2-3 times and load the lysate into the RNA column. Centrifuge for 30 s at 11,000 g. Place the column in a new 2 mL Collection Tube.
5. For the 1st wash, add 200 μL Buffer RAW2 to the NucleoSpin® RNA Columns. Centrifuge for 30 s at 11,000 x g. Place the column into a new 2 mL Collection Tube.
6. For the 2nd wash, add 600 μL Buffer RA3 to the NucleoSpin® RNA Columns. Centrifuge for 30 s at 11,000g. Discard the flowthrough and place the column back into the Collection Tube.
7. For the 3rd wash, add 250 μL of Buffer RA3 to the NucleoSpin® RNA Columns. Centrifuge for 2 min at 11,000g to dry the membrane completely. Place the column into a nuclease-free Collection Tube
8. Elute the RNA in 60 μL RNase-free H2O, and centrifuge at 11,000g for 1 min.
9. Store the RNA at -20°C until ready to use

## Results:

The tubes appear to be more transparent than the original tubes, indicating that this RNA had some level of purification.

Notes: *Since we were unsure if ethanol had already been added to the RA3 buffer from this kit, we substituted RNA wash buffer from the Zymo kit. We proceeded to run the miRNA on the nanodrop to determine the concentrations. The concentration of the miRNA is 9.8 ng/uL and the concentration of the crRNA is 25.6 ng/uL.*

Stop Time: 8:30 pm

Next Time: Test the components in the full Cas13 system to achieve proof of concept.

---

# Date: 9/24/23

Names: Erik Bergstrom, Danielle Dotson, Molly Ripper

Start Time: 2:30 PM

Purpose: To test our Cas13 system for initial functioning and proof of concept.

## Protocol

### Setup

* Use literature to find the concentrations of components needed for the experiment, and use C1V1 = C2V2 to determine the amount needed.
* Thaw components and divide both the miRNA and crRNA into separate 5 uL aliquots.
* Dilute Cas13 enzyme to 63.3 ug/mL by adding 1 uL of the enzyme to 14.8 uL of storage buffer (comprised of 0.3 M NaCl 20 mM Tris-HCl, ph 8.0 0.5 mM EDTA 1 mM DTT 50% Glycerol)

| Component | Starting Concentration | Amount |
| --- | --- | --- |
| Milli-Q Water |  | **3.5 ul** |
| crRNA | **25.6 ng/ul** | **0.5 ul** |
| Cas13 Enzyme | **63.3 ug/ul** | 2 ul |
| Cas13 reaction buffer (200 mM HEPES, 50 mM MgCl2, 1 M NaCl, 1 mM EDTA, pH 6.5) | **10 X** | **2 ul** |
| miRNA | **9.8 ng/ul** | **12 ul** |
| Total Volume: |  | 20 µL |

1. Add Milli-Q water, Cas13 reaction buffer, crRNA, and Cas13 in the order listed to a small Eppendorf tube
2. Incubate the solution at 37 C for 30 minutes in a water bath.
3. Remove the tube from the water bath and add 2 uL of miRNA to the solution and mix carefully by gently inverting the tube.
4. Incubate the solution again at 37 C for 5 minutes.

## Results:

Two tubes contain our system. The ideal outcome is that both tubes contain fragments of miRNA that have been cut by Cas13.

Stop Time: 4:20 pm

Next Steps: Run a gel electrophoresis experiment using ethidium bromide to determine if the miRNA was cut in the presence of Cas13.

---

# Date: 9/24/23

Names: Molly Ripper, Erik Bergstrom, Danielle Dotson

Start Time: 3:30 PM

Purpose: To run the gel electrophoresis to determine if miRNA-424-5p was cut in the presence of Cas13 and the guide RNA.

## Protocol:

1. Measure 0.5 g agarose powder and add it to a 250 mL flask.
2. Add 50 mL 1x TAE buffer to the flask.
3. Dissolve the agarose in a microwave or hot (>95°C) water bath until the solution becomes clear. If using a microwave, heat the solution for several short intervals. The agarose is completely dissolved when the solution is completely clear.
4. Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. The solution is cool enough when you can grab the flask with your bare hand.
5. Add 2 μL of ethidium bromide to the solution and swirl to mix. Wear gloves when handling ethidium bromide and whenever handling the gel for the rest of the protocol.
6. Secure the rails on either side of the gel casting tray in the up position such that the gel will not leak out of the tray as it cools.
7. Pour the melted agarose solution into the casting tray. Place the comb in the comb slot near the end of the casting tray.
8. Let the gel cool until it is solid (10-20 minutes). It will become opaque as it cools.
9. Carefully unscrew and lower the rails on the gel box.
10. Place the gel in the electrophoresis chamber with the wells on the negative (black) end of the gel box. If the gel is placed in the wrong orientation, the DNA will run off the end of the gel and the sample will be lost!
11. Add enough TAE buffer so that the gel is completely submerged by at least 2-3 mm.
12. Carefully and slowly pull the comb straight up and out of the gel.
13. Prepare a chart to keep track of which sample you will place in each well.

    1. Well 1 - Ladder
    2. Well 2 - miRNA
    3. Well 3 - Tube 3-1
    4. Well 4 - Tube 3-2
14. Add 2 μL loading dye per 10 μL sample and mix thoroughly by pipetting up and down.
15. Add 10 μL of the ladder to an empty well.
16. Add 10 μL of each sample to a well using one sample per well, exhibiting caution.
17. Run the gel immediately so that the RNA samples do not diffuse out of the wells.
18. Put the gel on a UV illuminator to see the results.

## Results

![](https://static.igem.wiki/teams/4856/wiki/lab/lab1.png)

The gel only showed one band in the ladder lane. The three other lanes had no bands at all, but the loading dye could be seen.

Stop Time: 5:30 PM

Next Time: Repeating the experiment with a new method to detect cut miRNA.

---

# Date: 10/7/23

Names: Erik Bergstrom

Start Time: 1:30 PM

Purpose: To test our Cas13 system for initial functioning and proof of concept.

## Protocol:

### Setup:

| Component | Starting Concentration | Amount |
| --- | --- | --- |
| Milli-Q Water |  | **3.5 ul** |
| crRNA | **25.6 ng/ul** | **0.5 ul** |
| Cas13 Enzyme | **63.3 ug/ul** | 2 ul |
| Cas13 reaction buffer (200 mM HEPES, 50 mM MgCl2, 1 M NaCl, 1 mM EDTA, pH 6.5) | **10 X** | **2 ul** |
| miRNA | **19.7 ng/ul** | **12 ul** |
| Total Volume: |  | 20 µL |

23. Add Milli-Q water, Cas13 reaction buffer, crRNA, and Cas13 in the order listed to a small Eppendorf tube.
24. Incubate the solution at 37 C for 1 hour in a water bath.
25. Remove the tube from the water bath and add 2 uL of miRNA to the solution and mix carefully by gently inverting the tube.
26. Incubate the solution again at 37 C for 10 minutes.

Stop Time: 3:45 pm

Next Time: Run this result in a gel to examine if the Cas13 cut the miRNA.

---

# Date: 10/7/23

Names: Erik Bergstrom

Start Time: 3:45 PM

Purpose: To run the gel electrophoresis to determine if miRNA-424-5p was cut in the presence of Cas13 and the guide RNA.

## Protocol:

1. Measure 2 g agarose powder and add it to a 250 mL flask.
2. Add 50 mL 1x TAE buffer to the flask.
3. Dissolve the agarose in a microwave or hot (>95°C) water bath until the solution becomes clear. If using a microwave, heat the solution for several short intervals. The agarose is completely dissolved when the solution is completely clear.
4. Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. The solution is cool enough when you can grab the flask with your bare hand.
5. Add 2 μL of ethidium bromide to the solution and swirl to mix. Wear gloves when handling ethidium bromide and whenever handling the gel for the rest of the protocol.
6. Secure the rails on either side of the gel casting tray in the up position such that the gel will not leak out of the tray as it cools.
7. Pour the melted agarose solution into the casting tray. Place the comb in the comb slot near the end of the casting tray.
8. Let the gel cool until it is solid (10-20 minutes). It will become opaque as it cools.
9. Carefully unscrew and lower the rails on the gel box.
10. Place the gel in the electrophoresis chamber with the wells on the negative (black) end of the gel box. Add enough TAE buffer so that the gel is completely submerged by at least 2-3 mm.
11. Carefully and slowly pull the comb straight up and out of the gel.
12. Prepare a chart to keep track of which sample you will place in each well.

    1. Well 1 - Reaction mixture from the previous experiment
    2. Well 2 - miRNA (12 uL total miRNA)
13. Add 2 μL loading dye per 10 μL sample and mix thoroughly by pipetting up and down.
14. Add 10 μL of each sample to a well using one sample per well, exhibiting caution.
15. Run the gel immediately so that the RNA samples do not diffuse out of the wells.
16. Put the gel on a UV illuminator to see the results.

## Results:

![](https://static.igem.wiki/teams/4856/wiki/lab/lab2.jpg)

Stop Time: 5:30 pm

Next Steps: Running the gel with crRNA and miRNA combined as an additional control and a ladder would be a better validation of our protocol. Only one control lane was used in this experiment due to the team having insufficient miRNA for a second control.

---

# Date: 10/8/2023

Names: Erik Bergstrom

Start Time: 4:30 pm

Purpose: In Vitro transcription to produce miRNA-424-5p and other miRNA

## [Protocol:](https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0012652_TranscriptAid_T7_High_Yield_Transcription_UG.pdf)

### Set-Up

* Added MilliQ water to DNA from IDT for miR-16, miR-145, miR-10b, miR21 to suspend in water at 4 ug/uL

  miR-16: 545 uL

  miR-145: 525 uL

  miR-10b: 605 uL

  miR-21: 525 uL
* All tubes result in a concentration of 4 µg/µL in the original DNA tube.
* Thaw all frozen reaction components, mix, and centrifuge briefly to collect all drops.
* Keep TranscriptAid Enzyme Mix and nucleotides on ice.
* Keep the 5X TranscriptAid Reaction Buffer at room temperature.
* Combine equal volumes of the four NTP solutions in one tube for convenience and reduction of pipetting steps

1. | Compoent | Amount |
   | --- | --- |
   | DEPC-treated water | to 20 µL → **5.5 µL** |
   | 5X TranscriptAid Reaction Buffer | **4 µL** |
   | ATP/CTP/GTP/UTP mix\* | **8 µL**b |
   | Template DNA | 2 µg → **0.5 µL** |
   | TranscriptAid Enzyme Mix | **2 µL** |
   | Total volume: | 20 µL |

   \* Equal volumes of the four provided NTP solutions combined in one tube.

   Tubes: miRNA 424, miRNA 424 10/8, miRNA 16, miRNA 145, miRNA 10b, miRNA 21
2. Mix thoroughly, spin briefly to collect all drops, and incubate at 37°C for 4-8 hours (short transcript).

Stop Time: 9:00 PM

## Results:

Several tubes of RNA were prepared that needed to be purified.

The concentrations of the RNA were:

| Sample | Concentration (ng/uL) |
| --- | --- |
| miR-424-1 | 4.7 |
| miR-424-2 | 5.1 |
| miR-424-3 | 6.8 |
| miR-424-4 | 6.6 |
| miR-424-5 | 3.3 |
| miR-424-6 | 4.1 |
| miR-10b | 8.4 |
| miR-16 | 7.9 |
| miR-21 | 8.4 |
| miR-145 | 6.2 |

---

# Date: 10/9/2023

Names: Danielle Dotson, Matthew Gaston, Chambre Garcia

Start Time: 6:00 pm

Purpose: RNA Purification of transcription products from the last experiment

## [Protocol:](https://www.mn-net.com/media/pdf/b0/51/ee/Instruction-NucleoSpin-RNA.pdf)

1. Add RNase-free H2O to each of our 20 µL tubes of product from the in vitro transcription experiment up to 100 µL.
2. In six 1.5 ml centrifuge tubes, prepare a Buffer RA1/ethanol premix with a ratio of 1:1. 300 μL Buffer RA1 and 300 μL ethanol (96–100 %) were mixed for each 100 μL RNA sample.
3. Add 600μL of Buffer RA1/ethanol premix to 100µL of sample RNA and vortex to mix
4. A NucleoSpin® RNA Column was placed in a collection tube. Pipette the lysate up and down 2-3 times and load lysate into RNA column. Centrifuge for 30 s at 11,000 g. Place the column in a new 2 mL Collection Tube.
5. For the 1st wash, add 200 μL Buffer RAW2 to the NucleoSpin® RNA Columns. Centrifuge for 30 s at 11,000 x g. Place the column into a new 2 mL Collection Tube.
6. For the 2nd wash, add 600 μL Buffer RA3 to the NucleoSpin® RNA Columns. Centrifuge for 30 s at 11,000g. Discard flowthrough and place the column back into the Collection Tube.
7. For the 3rd wash, add 250 μL of Buffer RA3 to the NucleoSpin® RNA Columns. Centrifuge for 2 min at 11,000g to dry the membrane completely. Place the column into a nuclease free Collection Tube
8. Elute the RNA in 60 μL RNase-free H2O, and centrifuge at 11,000g for 1 min.
9. Store the RNA at -20°C until ready to use

## Results:

Nanodrop results show a concentration of 22.7 ng/uL for miRNA-145, 28.4 ng/uL for miRNA 424, 12 ng/uL for miRNA-424 (10/8), 26.2 ng/uL for miRNA-16, 17.5 ng/uL for miRNA-21, and 6.2 ng/uL for miRNA-10b.

Notes: *Since we were unsure if ethanol had already been added to the RA3 buffer from this kit, we substituted RNA wash buffer from the Zymo kit.*

Stop Time: 8:30 pm

Next Time: Test the different miRNA with Cas13 to determine the specificity of the enzyme.

---

# Date: 10/10/23

Names: Danielle Dotson, Molly Ripper

Start Time: 1:00 PM

Purpose: To test our Cas13 system for initial functioning and proof of concept using a more informative gel.

## Protocol:

### Set-up

| Component | Starting Concentration | Amount |
| --- | --- | --- |
| Milli-Q Water |  | **6.5 ul** |
| crRNA | **25.6 ng/ul** | **0.5 ul** |
| Cas13 Enzyme | **63.3 ug/ul** | 2 ul |
| Cas13 reaction buffer (200 mM HEPES, 50 mM MgCl2, 1 M NaCl, 1 mM EDTA, pH 6.5) | **10 X** | **2 ul** |
| miRNA | **28.4 ng/ul** | **9 ul** |
| Total Volume: |  | 20 µL |

1. Label 7 tubes as shown below:

   | miRNA | crRNA | Cas13 | miRNA + crRNA | miRNA + CAS13 | crRNA + CAS13 | miRNA + crRNA + CAS13 |
   | --- | --- | --- | --- | --- | --- | --- |

   All tubes will have Milli-Q water and reaction buffer. The labels above indicate what will be added to each tube for the reaction.
2. Add Milli-Q water, Cas13 reaction buffer, crRNA, and Cas13 in the order listed to the appropriate Eppendorf tube.
3. Incubate the solution at 37 C for 1 hour in a water bath.
4. Remove the tube from the water bath and add 9 uL of miRNA to the solution as well as enough Milli-Q to bring it to 20 uL and mix carefully by gently inverting the tube.
5. Incubate the solution again at 37 C for 10 minutes.

Stop Time: 3:45 pm

Next Time: Run this result in a gel to examine if the Cas13 cut the miRNA.

---

# Date: 10/10/23

Names: Molly Ripper, Danielle Dotson

Start Time: 1:30 PM

Purpose: To rerun the gel electrophoresis to determine if miRNA-424-5p was cut in the presence of Cas13 and the guide RNA. Includes more bands as controls to make result interpretation easier.

## Protocol:

1. Measure 2 g agarose powder and add it to a 250 mL flask.
2. Add 50 mL 1x TAE buffer to the flask.
3. Dissolve the agarose in a microwave or hot (>95°C) water bath until the solution becomes clear. If using a microwave, heat the solution for several short intervals. The agarose is completely dissolved when the solution is completely clear.
4. Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. The solution is cool enough when you can grab the flask with your bare hand.
5. Add 2 μL of ethidium bromide to the solution and swirl to mix. Wear gloves when handling ethidium bromide and whenever handling the gel for the rest of the protocol.
6. Secure the rails on either side of the gel casting tray in the up position such that the gel will not leak out of the tray as it cools.
7. Pour the melted agarose solution into the casting tray. Place the comb in the comb slot near the end of the casting tray.
8. Let the gel cool until it is solid (10-20 minutes). It will become opaque as it cools.
9. Carefully unscrew and lower the rails on the gel box.
10. Place the gel in the electrophoresis chamber with the wells on the negative (black) end of the gel box. Add enough TAE buffer so that the gel is completely submerged by at least 2-3 mm.
11. Carefully and slowly pull the comb straight up and out of the gel.
12. Prepare a chart to keep track of which sample you will place in each well.

    | RNA Ladder | purified crRNA | Purified miRNA | CAS13 | crRNA + CAS13 | miRNA + CAS13 | miNA + crRNA | miRNA + crRNA + CAS13 |
    | --- | --- | --- | --- | --- | --- | --- | --- |
13. Add 2 μL loading dye per 10 μL sample and mix thoroughly by pipetting up and down.
14. Before adding to the gel, the samples must be denatured to prevent the formation of secondary structures.
15. Incubate in a 70°C water bath for 10 minutes.
16. Chill samples on ice for 3 minutes and spin briefly.
17. Add 10 μL of each sample to a well using one sample per well, exhibiting caution.
18. Run the gel immediately so that the RNA samples do not diffuse out of the wells.
19. Put the gel on a UV illuminator to see the results.

## Results

![](https://static.igem.wiki/teams/4856/wiki/lab/lab3.png)

Stop Time: 5:10 PM

Next Steps: Troubleshooting our gel.

---

# Date: 10/10/23

Names: Molly Ripper, Danielle Dotson, Erik Bergstrom, Austin Thrasher

Start Time: 7:30 PM

Purpose: To rerun the gel electrophoresis to determine if miRNA-424-5p was cut in the presence of Cas13 and the guide RNA. Includes more bands as controls to make result interpretation easier.

## Protocol:

1. Measure 2 g agarose powder and add it to a 250 mL flask.
2. Add 50 mL 1x TAE buffer to the flask.
3. Dissolve the agarose in a microwave or hot (>95°C) water bath until the solution becomes clear. If using a microwave, heat the solution for several short intervals. The agarose is completely dissolved when the solution is completely clear.
4. Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. The solution is cool enough when you can grab the flask with your bare hand.
5. Add 2 μL of ethidium bromide to the solution and swirl to mix. Wear gloves when handling ethidium bromide and whenever handling the gel for the rest of the protocol.
6. Secure the rails on either side of the gel casting tray in the up position such that the gel will not leak out of the tray as it cools.
7. Pour the melted agarose solution into the casting tray. Place the comb in the comb slot near the end of the casting tray.
8. Let the gel cool until it is solid (10-20 minutes). It will become opaque as it cools.
9. Carefully unscrew and lower the rails on the gel box.
10. Place the gel in the electrophoresis chamber with the wells on the negative (black) end of the gel box. Add enough TAE buffer so that the gel is completely submerged by at least 2-3 mm.
11. 5 ul of ethidium bromide was added to the positive side of the electrophoresis tray to help avoid the EtBr running off of the gel.
12. Carefully and slowly pull the comb straight up and out of the gel.
13. Prepare a chart to keep track of which sample you will place in each well.

    | RNA Ladder (5 ul) | purified crRNA | Purified miRNA | CAS13 | crRNA + CAS13 | miRNA + CAS13 | miNA + crRNA | miRNA + crRNA + CAS13 |
    | --- | --- | --- | --- | --- | --- | --- | --- |
14. Add 10 μL 2x RNA loading dye per 10 μL sample and mix thoroughly by pipetting up and down.
15. Add 20 μL of each sample to a well using one sample per well, exhibiting caution.
16. Run the gel immediately so that the RNA samples do not diffuse out of the wells.
17. Put the gel on a UV illuminator to see the results.
18. The ethidium bromide had ran off of the gel again

## Results:

High contrast image

![](https://static.igem.wiki/teams/4856/wiki/lab/lab4.png)

Low contrast image

![](https://static.igem.wiki/teams/4856/wiki/lab/lab5.png)

Stop time: 9:00 PM

Next steps: Run the experiment again and attempt to visualize results on another gel. Try to denature the miRNA to ensure the miRNA runs on the gel properly.

---

# Date: 10/11/2023

Names: Erik Bergstrom, Star Nevded, Molly Ripper

Start Time: 11:30 AM

Purpose: To test our Cas13 system for initial functioning and proof of concept using a more informative gel. Also testing the effect of other miRNA’s on the system.

## Protocol:

### Set-up

| Component | Starting Concentration | Amount |
| --- | --- | --- |
| Milli-Q Water |  | **To 20 uL** |
| crRNA | **25.6 ng/ul** | **0.5 ul** |
| Cas13 Enzyme | **63.3 ug/ul** | 2 ul |
| Cas13 reaction buffer (200 mM HEPES, 50 mM MgCl2, 1 M NaCl, 1 mM EDTA, pH 6.5) | **10 X** | **2 ul** |
| miRNA-424-5p | **28.4 ng/ul** | **9 ul** |
| miRNA-10b | 8.4 ng/ul | 10 uL |
| miRNA-21 | 8.4 ng/ul | 10 uL |
| Total Volume: |  | 20 µL |

1. |  | miRNA | crRNA | Cas13 | miRNA + crRNA | miRNA + CAS13 | crRNA + CAS13 | miRNA + crRNA + CAS13 | miRNA-10b + crRNA + CAS13 | miRNA-21 + crRNA + CAS13 |
   | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
   | miRNA | 9 | 0 | 0 | 9 | 9 | 0 | 9 | 10 | 10 |
   | crRNA | 0 | 0.5 | 0 | 0.5 | 0 | 0.5 | 0.5 | 0.5 | 0.5 |
   | CAS13 | 0 | 0 | 2 | 0 | 2 | 2 | 2 | 2 | 2 |
   | buffer | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
   | water | 9 | 17.5 | 16 | 8.5 | 7 | 15.5 | 6.5 | 5.5 | 5.5 |

   All tubes will have Milli-Q water and cas reaction buffer. The labels above indicate what will be added to each tube for the reaction.
2. Add Milli-Q water, Cas13 reaction buffer, crRNA, and Cas13 in the order listed to the appropriate Eppendorf tube.
3. Incubate the solution at 37 C for 1 hour using a PCR machine
4. Remove the tube from the water bath and add 9 uL of miRNA to the solution, mix by inverting the tube
5. Incubate the solution again at 37 C for 10 minutes.

## Results:

Completed reactions ready to run on a gel

Stop time: 2:00 PM

Next steps: Running the solutions on a gel.

---

# Date: 10/11/23

Names: Erik Bergstrom, Star Nevded

Start Time: 2:30 PM

Purpose: To rerun the gel electrophoresis to determine if miRNA-424-5p was cut in the presence of Cas13 and the guide RNA. Includes more bands as controls to make result interpretation easier, and to test if RNA cutting, if any, is specific. Addition of a denature step.

## Protocol:

1. Measure 2 g agarose powder and add it to a 250 mL flask.
2. Add 50 mL 1x TAE buffer to the flask.
3. Dissolve the agarose in a microwave or hot (>95°C) water bath until the solution becomes clear. If using a microwave, heat the solution for several short intervals. The agarose is completely dissolved when the solution is completely clear.
4. Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. The solution is cool enough when you can grab the flask with your bare hand.
5. Add 2 μL of ethidium bromide to the solution and swirl to mix. Wear gloves when handling ethidium bromide and whenever handling the gel for the rest of the protocol.
6. Secure the rails on either side of the gel casting tray in the up position such that the gel will not leak out of the tray as it cools.
7. Pour the melted agarose solution into the casting tray. Place the comb in the comb slot near the end of the casting tray.
8. Let the gel cool until it is solid (10-20 minutes). It will become opaque as it cools.
9. Carefully unscrew and lower the rails on the gel box.
10. Place the gel in the electrophoresis chamber with the wells on the negative (black) end of the gel box. Add enough TAE buffer so that the gel is completely submerged by at least 2-3 mm.
11. Carefully and slowly pull the comb straight up and out of the gel.
12. Prepare a chart to keep track of which sample you will place in each well.

    | RNA Ladder (5 ul) | purified crRNA | Purified miRNA | CAS13 | crRNA + CAS13 | miRNA + CAS13 | miNA + crRNA | miRNA + crRNA + CAS13 |
    | --- | --- | --- | --- | --- | --- | --- | --- |
13. Add 10 μL 2x RNA loading dye per 10 μL sample and mix thoroughly by pipetting up and down.
14. Before adding to the gel, the samples must be denatured to prevent the formation of secondary structures.
15. Incubate in a 70°C water bath for 10 minutes.
16. Chill samples on ice for 3 minutes and spin briefly.
17. 5 ul of ethidium bromide was added to the positive side of the electrophoresis tray to help avoid the issue with the EtBr running off the negative end of the gel.
18. Add 20 μL of each sample to a well using one sample per well, exhibiting caution.
19. Run the gel immediately so that the RNA samples do not diffuse out of the wells.
20. Put the gel on a UV illuminator to see the results.
21. The gel was allowed to soak in TAE buffer with EtBr for 40 minutes after being imaged initially
22. The gel was imaged again on an UV illuminator

## Results:

First Set of Images

![](https://static.igem.wiki/teams/4856/wiki/lab/lab6.jpg)
![](https://static.igem.wiki/teams/4856/wiki/lab/lab7.jpg)

Second Set of Images

![](https://static.igem.wiki/teams/4856/wiki/lab/lab8.jpg)
![](https://static.igem.wiki/teams/4856/wiki/lab/lab9.jpg)

Stop time: 4:00 pm

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

# Engineering Success

---

Our engineering success came from the initial proof of concept.
Our team has spent over 3 months researching everything to know about CRISPR systems and how the Cas enzyme works.
Specifically, we were concerned about the activation mechanism of the RNase activity since there were not many articles directly referring to the proposed mechanism, or much information from protocols and how it works.
Given the complexity of the system, we decided to start with a proof of concept experiment to showcase our theories from compiled research.

---

## Design Cycle

---

### Design:

We designed our components based on a combination of multiple papers found for CRISPR-Cas13 systems.
The miRNA used was miRNA 424-5p, which was previously sequenced in a research article.
The crRNA was designed to be a complement to both the miRNA and the Cas13 enzyme.
It has a section that makes a stem-loop structure to bind to the Cas13 as well as a section that base pairs with the miRNA.
The theory is that the crRNA binds to the Cas13 as a precursor to the activation of RNase activity.
When the miRNA is added to the system, it base pairs with the crRNA to activate the Cas13 enzyme.
This enzyme will then begin cutting in the system, which is a goal for our product later on.

### Build:

We ordered miRNA-424-5p and the specific crRNA from the design phase above.
We also ordered Cas13a for our initial proof of concept experiment.
Using multiple protocols and research, we developed a protocol that binds the Cas13 to the crRNA in an incubation step.
It then adds miRNA and allows it to bind to the crRNA for the activation of RNase activity, also in an incubation step.

### Test:

In order to test if the proof of concept is sound, we would use gel electrophoresis to compare pure miRNA and the results from the CRISPR
system to see if the miRNA was cut. If the gel shows many small bands in the CRISPR column and one band in the miRNA column,
it can be concluded that the proof of concept is valid. The CRISPR system will cut samples when miRNA is present.
A follow-up test would be added where a random RNA that is not similar to the miRNA is put in a sample with Cas13 and the crRNA and compared to the
designed CRISPR system. Both samples would be put into a gel again to be compared to the uncut miRNA and RNA.
We would expect the designed system to show cut miRNA and the sample with the random RNA to show uncut RNA bands.

### Learn:

The gel electrophoresis showed only a band from the ladder. We learned that a 1% agarose gel created “holes” too big that allowed our 22 nucleotide-long chain to flow through and disassociate into the buffer solution. After further discussion, it was concluded that a new method is needed to validate that miRNA is cut by Cas13. The new method includes a protocol for gel electrophoresis, but this one is specifically meant for RNA. This then would allow the team to ensure that the miRNA is cut into small fragments when in a solution with the Cas13 enzyme and crRNA.
Our 4% agarose gel electrophoresis was a step in the correct direction, as we were able to see bands in the gel. The samples however were not denatured which caused an appearance of double bands for one component. The next gel would add a heat denaturation step in order to avoid a secondary structure formation. We also learned that we would want to add more lanes for the individual components as well using a RNA ladder for size measurements.
The next gel was able to give us more information, however we were unable to read half of it. Some of this may be due to too much ladder and running the gel for too long so the EtBr ran off.
We were able to modify this error by adding more EtBr to the cathode side to prevent it from running off of the gel.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

# Experiments

---

We utilized public databases such as NCBI, the National Institute of Health, and the National Library of Medicine to search for papers and design protocols; identified biomarkers by utilizing these papers to find RNA biomarkers commonly found in breast and other cancers; and searched for our RNA sequences using RNAcentral. We selected several other variants of miRNA such as miR-320c, miR-10b-5p, miR-21, and miR-16 to test because these miRNAs are either prominent miRNAs found in urine or have been identified as potentially useful for breast cancer diagnosis. To confirm the results of our experiment we decided to use electrophoresis to detect miRNA bands within our sample. This will test whether or not they had been cut by our Cas13 enzyme or not.

Our first protocol was in-vitro transcription to produce the miRNA and crRNA we would use for our experiments. We used a T7 TranscriptAid kit for the in vitro transcription.

## [Protocol:](https://www.thermofisher.com/document-connect/document-connect.html?url=https://assets.thermofisher.com/TFS-Assets%2FLSG%2Fmanuals%2FMAN0012652_TranscriptAid_T7_High_Yield_Transcription_UG.pdf)

(page 6)

### Set-Up

* Added 1125 µL of MilliQ water to 2023MissouriMinersT7CRRNA424 (crRNA). Results in a concentration of 4 µg/µL in the original DNA tube.
* Added 615 µL of MilliQ water to 2023MissouriMinersT7MIRNA424 (miRNA). Results in a concentration of 4 µg/µL in the original DNA tube.
* Thaw all frozen reaction components, mix, and centrifuge briefly to collect all drops.
* Keep TranscriptAid Enzyme Mix and nucleotides on ice.
* Keep the 5X TranscriptAid Reaction Buffer at room temperature.
* Combine equal volumes of the four NTP solutions in one tube for convenience and reduction of pipetting steps

1. Combine the following reaction components at room temperature in the order given:

   | Compoent | Amount |
   | --- | --- |
   | DEPC-treated water | to 20 µL → **5.5 µL** |
   | 5X TranscriptAid Reaction Buffer | **4 µL** |
   | ATP/CTP/GTP/UTP mix\* | **8 µL**b |
   | Template DNA | 2 µg → **0.5 µL** |
   | TranscriptAid Enzyme Mix | **2 µL** |
   | Total volume: | 20 µL |

   \* Equal volumes of the four provided NTP solutions combined in one tube.

    Tubes: (miRNA, trans, 4/20) and (crRNA, trans, 4/20)
2. Mix thoroughly, spin briefly to collect all drops, and incubate at 37°C for 4-8 hours (short transcript).

---

Our second experiment’s purpose was to purify the transcription products from our first experiment. For this experiment, we utilized Nucleospin and a centrifuge to separate the transcription products from the rest of the solution. The tubes appeared more transparent than initially, indicating some level of purification.

## [Protocol:](https://www.mn-net.com/media/pdf/b0/51/ee/Instruction-NucleoSpin-RNA.pdf)

1. Add RNase-free H2O to each of our 20 µL tubes of product from the in vitro transcription experiments up to 100 µL.
2. In two 1.5 ml centrifuge tubes, prepare a Buffer RA1/ethanol premix with a ratio of 1:1. 300 μL Buffer RA1 and 300 μL ethanol (96–100 %) were mixed for each 100 μL RNA sample.
3. Add 600μL of Buffer RA1/ethanol premix to 100µL of sample RNA and vortex to mix
4. A NucleoSpin® RNA Column was placed in a collection tube. Pipette the lysate up and down 2-3 times and load the lysate into the RNA column. Centrifuge for 30 s at 11,000 g. Place the column in a new 2 mL Collection Tube.
5. For the 1st wash, add 200 μL Buffer RAW2 to the NucleoSpin® RNA Columns. Centrifuge for 30 s at 11,000 x g. Place the column into a new 2 mL Collection Tube.
6. For the 2nd wash, add 600 μL Buffer RA3 to the NucleoSpin® RNA Columns. Centrifuge for 30 s at 11,000g. Discard the flowthrough and place the column back into the Collection Tube.
7. For the 3rd wash, add 250 μL of Buffer RA3 to the NucleoSpin® RNA Columns. Centrifuge for 2 min at 11,000g to dry the membrane completely. Place the column into a nuclease-free Collection Tube
8. Elute the RNA in 60 μL RNase-free H2O, and centrifuge at 11,000g for 1 min.
9. Store the RNA at -20°C until ready to use

We measured the concentrations of RNA on a nanodrop and found that the final concentration of crRNA was 25.6 ng/ul, and the final concentration of miRNA was 8.8 ng/ul.

---

The third experiment tested if the target sequence was cut. For the protocol, we utilized electrophoresis to attempt to detect if our Cas13a enzyme had cut the miRNA. We were unable to visualize the miRNA in our gel. From these results, we realized we needed a more sophisticated gel and a higher concentration of RNA to better see the bands. Further testing revolved around creating a gel electrophoresis protocol that would allow us to visualize the bands of very small ( <20 nt ) degraded miRNA.

## Protocol for Reaction tubes:

| Component | Starting Concentration | Amount |
| --- | --- | --- |
| Milli-Q Water |  | **6.5 ul** |
| crRNA | **25.6 ng/ul** | **0.5 ul** |
| Cas13 Enzyme | **63.3 ug/ul** | 2 ul |
| Cas13 reaction buffer (200 mM HEPES, 50 mM MgCl2, 1 M NaCl, 1 mM EDTA, pH 6.5) | **10 X** | **2 ul** |
| miRNA | **28.4 ng/ul** | **9 ul** |
| Total Volume: |  | 20 µL |

1. Label 7 tubes as shown below:

   | miRNA | crRNA | Cas13 | miRNA + crRNA | miRNA + CAS13 | crRNA + CAS13 | miRNA + crRNA + CAS13 |
   | --- | --- | --- | --- | --- | --- | --- |

   All tubes will have Milli-Q water and reaction buffer. The labels above indicate what will be added to each tube for the reaction.
2. Add Milli-Q water, Cas13 reaction buffer, crRNA, and Cas13 in the order listed to the appropriate Eppendorf tube.
3. Incubate the solution at 37 C for 1 hour in a water bath.
4. Remove the tube from the water bath and add 9 uL of miRNA to the solution as well as enough Milli-Q to bring it to 20 uL and mix carefully by gently inverting the tube.
5. Incubate the solution again at 37 C for 10 minutes.

## Protocol for Current Gel Electrophoresis Method:

1. Measure 2 g agarose powder and add it to a 250 mL flask.
2. Add 50 mL 1x TAE buffer to the flask.
3. Dissolve the agarose in a microwave or hot (>95°C) water bath until the solution becomes clear. If using a microwave, heat the solution for several short intervals. The agarose is completely dissolved when the solution is completely clear.
4. Let the solution cool to about 50-55°C, swirling the flask occasionally to cool evenly. The solution is cool enough when you can grab the flask with your bare hand.
5. Add 2 μL of ethidium bromide to the solution and swirl to mix. Wear gloves when handling ethidium bromide and whenever handling the gel for the rest of the protocol.
6. Secure the rails on either side of the gel casting tray in the up position such that the gel will not leak out of the tray as it cools.
7. Pour the melted agarose solution into the casting tray. Place the comb in the comb slot near the end of the casting tray.
8. Let the gel cool until it is solid (10-20 minutes). It will become opaque as it cools.
9. Carefully unscrew and lower the rails on the gel box.
10. Place the gel in the electrophoresis chamber with the wells on the negative (black) end of the gel box. Add enough TAE buffer so that the gel is completely submerged by at least 2-3 mm.
11. Carefully and slowly pull the comb straight up and out of the gel.
12. Prepare a chart to keep track of which sample you will place in each well.

    | RNA Ladder | purified crRNA | Purified miRNA | CAS13 | crRNA + CAS13 | miRNA + CAS13 | miNA + crRNA | miRNA + crRNA + CAS13 |
    | --- | --- | --- | --- | --- | --- | --- | --- |
13. Add 2 μL loading dye per 10 μL sample and mix thoroughly by pipetting up and down.
14. Before adding to the gel, the samples must be denatured to prevent the formation of secondary structures.
    1. Incubate in a 70°C water bath for 10 minutes.
    2. Chill samples on ice for 3 minutes and spin briefly.
15. Add 10 μL of each sample to a well using one sample per well, exhibiting caution.
16. Run the gel immediately so that the RNA samples do not diffuse out of the wells.
17. Put the gel on a UV illuminator to see the results.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

# Results

## Successful Results

* In vitro transcription of our crRNA and miRNA
* RNA purification of our crRNA and miRNA

## Unsuccessful Results

* Initial functioning and proof of concept experiments

  + We performed variations of this experiment 5 separate times.
  + We believe that our lack of results was due to our inability to visualize our results. We attempted several versions of gel electrophoresis, altering gel percentage, amount of ladder/sample, amount of ethidium bromide, and introduced a denaturing step for the microRNA. We tried adding excess ethidium bromide into the TAE at the cathode end, and we attempted to let the gel soak in the TAE/EtBr mixture for readable results. Despite all of our efforts, none were successful.
  + Because our product size was so small (the miRNA was ~20bp, and smaller if the Cas13 became catalytically active), these gels were unable to display the small molecules.
  + If we were able to replicate this experiment again, we would have changed the type of gel we used instead of another EtBr agarose. Due to the size and funding of our lab, we were unable to obtain the necessary supplies to run a polyacrylamide, formaldehyde, or another gel that is able to more accurately and easily display miRNA.
* Gel #1

  ![](https://static.igem.wiki/teams/4856/wiki/gels/gel1.png)

  Visible bands only in the DNA ladder lane. Our miRNA and experimental results are not visible.
* Gel #2

  ![](https://static.igem.wiki/teams/4856/wiki/gels/gel2.jpg)

  Well #1 contained our Cas13+crRNA+miRNA reaction; well #2 contained just miRNA. The reaction mixture showed a bit of smearing, indicating possibly positive results. However, we cannot confirm that the smearing is due to successful cutting. We decided to run it again, this time with several other controls to help visualize and interpret the results. We also decided to add a denaturing step to prevent the miRNA from forming secondary structures and not separating properly in the gel.
* Gel #3

  ![](https://static.igem.wiki/teams/4856/wiki/gels/gel3.png)

  8 of the 10 wells contained a product, with the first well being an RNA ladder. The ladder showed unsuccessful separating, indicating an issue with our gel or with excess ladder used. We also noticed the EtBr began running off the gel, cutting off visibility where we would see our results. We ran it again to correct the EtBr and ladder issues.
* Gel #4

  ![](https://static.igem.wiki/teams/4856/wiki/gels/gel4.png)

  Ladder separated successfully. No product visible in any other well. This gel also contained random splotches of illumination. We did not denature the product for this gel, so we ran the experiment one last time to include that step.
* Gel #5

  ![](https://static.igem.wiki/teams/4856/wiki/gels/gel5.jpg)

  Only visible bands in the ladder lane. We suspect that the gel ran for too long, causing our small product to run off the gel. If we had the time, we would run this again for a shorter amount of time.

## Future Directions

Due to the timing of our project, we were unable to complete any more experiments. However, we have several directions we want to go with our project.

* Our fourth experiment after concluding that the miRNA is degraded by Cas13, would have tested the specificity of the enzyme by also adding other miRNA sequences and comparing the results. This would ensure that the Cas13 will only activate in the presence of miR-424-5p and no other miRNA, lowering the probability of false positives. We attempted this experiment on our last gel, however the gel showed no meaningful results.
* At this point, we planned to introduce our quencher fluorophore signal. When added to the experiment, the bond holding the two molecules together would be cleaved by catalytically active Cas13, causing the solution to fluoresce. This is the signal that would alert the consumer if miR-424-5p is detected in the urine.
* We discussed running this system with synthetic urine to test if other molecules in the urine would affect how the experiment runs.
* Lastly, once our system was proven to work, we aimed to test how our device would hold up when in a consumer’s possession. Several factors can impact the stability of our system, so we planned to test the effects of a few conditions: the number of freeze-thaw cycles, the storage temperature, the buffer solution used, and light vs. dark storage.

{% endblock %}