iGEM 2024 — Team missouri-miners

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# Lab Notebook

There was insufficient time to order parts or run wet lab experiments but if this project were to be continued then these would be the procedures most likely used.

NEB In vitro digestion of DNA with Cas9

Nuclease, S. pyogenes (M0386)

**Purpose:** To check for cas9 cutting and binding to target DNA in an in vitro enviroment.

## Protocol:

1. Assemble the reaction at room temperature in the following order:

   Nuclease-free water

   NEBuffer r3.1

   300 nM sgRNA 3

   1 µM Cas9 Nuclease, S. pyogenes (M0386S)

   Reaction volume

   20 µl

   3 µl

   µl (30 nM final)

   1 µl (~30 nM final)

   27 µl

   **Pre-incubate for 10 minutes at 25⁰C**

   30 nM substrate DNA

   Total reaction volume

   3 µl (3 nM final)

   30 µl
2. Mix thoroughly and pulse-spin in a microfuge.
3. Incubate at 37°C for 15 minutes.
4. Add 1 μl of Proteinase K to each sample, Mix thoroughly and pulse-spin in a microfuge.
5. Incubate at room temperature for 10 minutes.
6. Proceed with fragment analysis.

**Notes:** N/A

**Next:** Plasmid miniprep to start in vivo tests.

## Plasmid Miniprep

**Purpose:** To extract plasmid DNA for in vivo tests

## Protocol:

1. Gently swirl the contents of the culture tube to resuspend the cells. Transfer to a 15 mL centrifuge tube and spin for 5 min to pellet the cells.
2. Label a 1.5 mL tube. Add 200uL solution 1, resuspend the cells, and pipet the cell suspension into the microfuge tube.
3. Close the caps and place the tubes in a centrifuge (remember to balance the centrifuge by putting two tubes opposite one another) and spin at maximum speed for 20 s. After the spin, you should see amber liquid and a white pellet.
4. Withdraw and discard the supernatant using a pipettor, being careful not to disturb the cell pellet. Discard the supernatant in a waste container.
5. Add 200 uL of Solution 1 to each tube and resuspend the cells by vortexing. It's very important that the cell suspension is homogenous and no clumps are visible. The cells are now resuspended in a buffered solution with RNase.
6. Add 200 uL of Solution 2 to each tube. Close the caps and mix the solutions by rapidly inverting them a few times. DO NOT VORTEX.
7. Add 200 uL of ice-cold Solution 3 to each tube. Close the caps and mix the solutions by rapidly inverting them a few times. A white precipitate will form.
8. Place the tubes in a centrifuge (balanced) and spin at maximum speed for 5 minutes. The precipitate will pellet along the side of the tube.
9. Transfer the supernatants into clean 1.5 mL tubes, being careful not to pick up any of the precipitate. Discard the tubes with the precipitate and KEEP the tubes with the supernatant.
10. To each tube of supernatant add an equal volume (about 600 uL) of isopropanol. Close the caps and mix vigorously. Let the tubes stand at room temperature for 2 minutes, place them, with their hinges pointing outward from the center, in a centrifuge (balanced) and spin at maximum speed for 5 minutes.
11. Carefully remove and discard the supernatant.
12. Add 200 uL of ice cold absolute ethanol to each tube and mix by inversion several times.
13. Spin the tubes at maximum speed in a centrifuge for 2-3 minutes (hinges out).
14. Carefully remove and discard the supernatant. Try to get as much out as possible without dislodging the pellet of plasmid DNA.
15. Leave tubes open to let ethanol evaporate. When the ethanol is gone add 20 uL of nuclease free water to dissolve the pellet. Pipet the 20 uL in and out, up the side of the tube to ensure that all of the plasmid DNA comes into contact with the TE buffer.
16. Store your miniprep DNA in the freezer.

**Notes:** N/A

**Next:** Insert custom sequence into the plasmids and insert plasmids into lambda phages.

Later experiments could also include testing transformed phages on resistant E. coli to test for success.

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Protocols from:

1. https://www.neb.com/en-us/protocols/2014/05/01/in-vitro-digestion-of-dna-with-cas9-nuclease-s-pyogenes-m0386
2. Modified from http://csmbio.csm.jmu.edu/biology/courses/bio480\_580/mblab/miniprep.html

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## Design. Build. Test. Learn. Repeat.

#### Miners Dig Deeper.

Our phage-deployed system approach aims to combat antibiotic resistance in bacteria using lambda phages, a Craspase system, and gasdermin proteins. The system is designed to detect antibiotic-resistant genes in bacteria and trigger cell death via pyroptosis in bacteria presenting genes that match our gRNA sequence. Pyroptosis kills the cells via activated caspases that cleave gasdermin proteins. The cleavage of the gasdermin proteins causes pores to form in the cell membrane, prompting the cell to swell and eventually burst. Our project was initially inspired by Proteus, a project created by McGill University in 2023, which focused on utilizing human gasdermins to target pancreatic cancer. Building from this, our project shifted focus to the use of bacterial gasdermins endogenous to E. coli to target ampicillin-resistance. We first considered using Craspase for a caspase cascade but reverted to the use of gasdermins due to specificity concerns. A caspase cascade could be initiated by a variety of environmental factors which would not have led to a system with the desired specificity. Various alternatives were explored, including mRNA versus highly specific protein cleavage and b-lactamase specific proteases. The final design incorporates a fusion protein of Runella gasdermin, Csx-30, and a Craspase system containing the Csx-30 specific protease Csx-29. The RNA that codes for these systems is to be delivered by lambda phages specific to E. coli. This innovative approach combines several key components to create a highly specific targeting system for combating antibiotic-resistant bacteria.

The components of the project prominently feature Craspase: a protease complex that cleaves target proteins when activated by specific RNA molecules, and gasdermin proteins, which form pores in bacterial membranes, leading to pyroptosis. The fusion protein combines Csx-30 and gasdermin proteins, which are cleaved upon detection of antibiotic-resistant genes by the Csx-29 protease. Modified bacteriophages serve as the delivery method to introduce the system into bacteria. Linker design proved crucial for holding gasdermin halves together based on comparison with human gasdermins. Specificity concerns were addressed by using Csx-30 linkers and engineered E. Coli specific lambda phages. Locating the correct linking area in bacterial gasdermin required comparison with human gasdermin studies, while potential issues with gRNA mutation and unintended protease activity were considered in the design process.

In the future, further experimentation to confirm the correct linking area in bacterial gasdermin and a potential focus on targeting ampicillin-resistant genes in bacteria in clinical settings will be continued project goals. Further refinement of specificity and activation mechanisms are necessary to prevent unintended cell death. The project methodology followed iterative design principles including planning, education, design, development, testing, and analysis. We utilized literature review and online modeling for concept testing and refinement and employed vector building programs for sequence comparison and linker design. This comprehensive approach, combining cutting-edge biotechnology with rigorous scientific methodology, positions the project as a promising avenue for addressing the critical issue of antibiotic resistance in bacteria.

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