Individual Final Project

CAD platform for programmable living CA

HTGAA 2026: Individual Final Project Report

SECTION 1: ABSTRACT

1. Provide an abstract/summary for your project. (minimum 150 words) • Should be a self-contained description of the project

Bacteria can compute spatial patterns using chemical signals, the same way cellular automata compute patterns using rules. ->My project would build the bridge between a design tool that lets you specify a spatial rule (“cells switch state when neighbour density exceeds a threshold”) and translates it into an actual genetic circuit one could build.

• Should contain a brief outline of:

• Significance

• Broad objectives

CAD platform for programmable living CA

• Hypotheses

Why: Biology is becoming programmable but lacks a lot of the design infrastructure for exploring spatial systems and it’s more than possible to build them

• Specific aims

+build a simulation engine that translates CA rules into genetic circuit specification (validated in a wet-lab environment, potentially via Opentrons)

+design a spatial design tool for engineering microbial consortia that exhibit programmable cellular automata behaviour.

• Methods to be employed • Use lay language (i.e., understandable by the general public) as much as possible

SECTION 2: PROJECT AIMS

2. Outline three aims of your final project (min. 3 sentences, at least one for each aim) • The first aim should be structured “The first aim of my final project is to [insert an achievable experimental goal that encompasses your project] by utilizing [insert protocols/tools/strategies you will use to achieve your goal]”

• State or link any methods/experimental protocols/OpenTrons protocols/DNA or protein designs/protein design tools or models/Twist orders you will use
• You will provide a detailed, step-by-step outline of how you will achieve your goal for the first aim in the experimental design portion of this assignment (i.e., in question 7)
• Feel free to run your goal by a TA

Aim 1 — Build a single sender/receiver CA unit Sender cells express LuxI → produce 3OC6-HSL (AHL signal) Receiver cells express LuxR + GFP reporter → fluoresce above AHL threshold Goal: demonstrate density-dependent state switching (the CA rule in biology)

• The second aim should be a medium-term aim that is a follow-up to your first aim and focused on goals beyond this class, building toward your third, visionary aim

• For example, your second aim may be to successfully execute a set of experimental protocols, solve a specific problem, or develop a specific technology building upon the goals of your first aim.

• The third aim should be a visionary, long-term aim

• Reveal how the larger goal of the project can be impactful
• Examples: challenging an existing paradigm or clinical practice, addressing a critical barrier to progress in the field, describing how you envision a new technology to change how a certain type of research is conducted

SECTION 3: BACKGROUND

3. Provide background research for your final project. This should describe the current state of knowledge related to your project and be a critical evaluation of the literature that identifies the gap in knowledge that this project will fill. Cite at least 2 peer-reviewed research papers. (min. 4 sentences)

4. Describe how your project is innovative (min. 3 sentences) • Examples of topics to discuss

• Novel applications, usage, or development of theoretical concepts, approaches, methodologies, instrumentation, and/or interventions
• How it challenges current theories, paradigms, or ways in which technology/biological tools are used
• How your project pushes the boundaries of synthetic biology

5. Briefly expand upon the significance of your final project. (min. 5 sentences) • Examples of topics to discuss

• How your project solves a pressing problem in the world
• Importance of the problem it solves or the critical barrier(s) to progress in the field that the proposed project addresses
• The ways in which it contributes to the larger society
• How the proposed project will improve scientific knowledge, technical capability, and/or clinical practice in one or more broad fields
• How the concepts, methods, technologies, treatments, services, or preventative interventions that drive this field will be changed if the proposed aims are achieved

6. Describe the bioethical considerations involved in your project. (min. 2 paragraphs) • First paragraph: Include what ethical implications are involved in your project. Try to suggest ethical the principle(s) you may apply (e.g. non-maleficence, justice)? • Second paragraph: Describe the measures that should be taken to ensure that your project is ethical (both in how the research is conducted and in its broader implications for society). You may wish to answer the following questions:

• What action(s) do you propose?
• What are potential unintended consequences of your proposed actions?
• What could you have wrong (e.g., incorrect assumptions and uncertainties)?
• What are alternatives to your proposed actions? • Note: in an NIH proposal, an ethics statement is used to describe the relevance of this research to public health

SECTION 4: EXPERIMENTAL DESIGN

7. Create a detailed experimental plan for your final project. Include a timeline for each part of your experimental plan (i.e., how long you would expect each step in your final project to take). (min. 15 lines/sentences—a numbered list is acceptable) • Include specific methods/tools/technologies/biological concepts for each part of the final project and analysis • This section will be used to determine whether the experiments are well designed, feasible, and likely to succeed in testing your hypothesis • Often this section is broken into discrete tasks/sub-aims • For each experiment and/or analysis, include a description of your expected results • If possible, include figure(s) that visually shows a broad workflow of your project or a specific aspect of your experimental plan

SECTION 5: TECHNIQUES, TOOLS, AND TECHNOLOGY

8. We discussed and practiced various techniques related to synthetic biology throughout the semester. Place a check next to the techniques relevant to your project. Pipetting Pipetting Lab Safety Bioethical Considerations (must check this box)

DNA Gel Art DNA Sequencing DNA Editing (e.g., CRISPR) DNA Construct Design Restriction Enzyme Digestion Gel Electrophoresis DNA Purification From Gel Databases (e.g., GenBank, NCBI, Ensembl, and UCSC Genome Browser)

Opentrons Creating Code for Laboratory Automation PyLabRobot Using Liquid Handling Robots (e.g., Opentrons)

Protein Design Protein Design Models and Notebooks Databases Tools BioProduction BioProduction Chassis Selection (e.g., Dh5alpha) Registry of Standard Biological Parts FreeGenes Plasmid Preparation Bacterial Culturing Quality Control/Analysis Bacterial Processing (e.g., Centrifugation, Lysis, DNA Purification)

Cell Free Cell Free Reactions Freeze-Dried Cell Free Systems miniPCR Tools

Week 7: Gibson Assembly Primer Design or Selection PCR Reactions Gibson Assembly Other Cloning Methods (e.g., Restriction Enzyme Digestion or Gateway Cloning)

Week 8-9: CRISPR CRISPR/Cas9 Designing Prime Editing gRNA Creating Twist Order

9. Expand upon two techniques you checked in the previous question by describing how you would utilize those techniques in your final project. (min. 4 sentences)

SECTION 6: PROJECT VALIDATION

10. You are required to validate at least one aspect of your final project aims. This is to ensure that you are able to successfully apply a relevant synthetic biology technique to your project. Include figures if you have them—accuracy is critical in figures, tables, and graphs.

Here is a non-exhaustive list of acceptable validations: • Performing a PCR reaction using primers relevant to your final project • Performing a Gibson assembly relevant to your final project • Designing DNA relevant to your final project • Creating and performing a cell-free assay related to your final project • Creating and running code to validate an aspect of your final project • Developing a model or completing a computational analysis relevant to your project • Designing DNA construct(s) that can express at least one gene of interest, ordering it (via Twist), and testing of the expression of the construct(s) (potentially using an OpenTrons robot)

10a. What aspect of your final project did you choose to validate? (min. 2 sentences)

10b. Write down a detailed protocol of how you validated this aspect of your final project. (Numbered list or paragraph is fine)

DAY 0 (Prep stocks + plates)

use: LB broth: 100 mL agar plates: 4 chloramphenicol: 100 µL stock used DMSO: 100 µL AHL: one aliquot PBS: none yet

to do:

• pour 4 LB chloramphenicol agar plates
• make 100 mL LB + chloramphenicol
• prepare 1 mL chloramphenicol stock
• dissolve AHL to 10 mM in 100 µL DMSO
• label OT-2 plates

DAY 1 — Transform sender + receiver

use competent cells: 2 × 50 µL LB recovery: 2 mL plates: 2 chloramphenicol plates from Day 0

to do: Transform: sender → 1 tube receiver → 1 tube Plate both.

DAY 2 — Pick colonies + overnight cultures

use: LB broth: 15 mL chloramphenicol: 15 µL from stock

do: Pick - > grow 1 sender colony - > 5 mL sender 1 receiver colony - > 10 mL receiver

**DAY 3 — Freezer stocks + AHL curve **

use glycerol: 2 mL black plate: 1 receiver culture: 10 mL PBS: 10 mL AHL stock: 10 µL DMSO: already made stock

do freezer stocks 500 µL culture + 500 µL 50% glycerol sender + receiver OT-2 AHL curve Use 1 full 96-well plate Receiver in all wells.

DAY 4 — Sender overnight culture

use LB broth: 10 mL chloramphenicol: 10 µL

do Grow sender: 10 mL LB + chlor for supernatant assay.

DAY 5 — Sender → receiver assay

use black plate: 1 sender supernatant: 5 mL receiver culture: 10 mL PBS: 10 mL

do Spin sender. Collect supernatant. OT-2: sender supernatant dilution receiver in all wells GFP time-lapse

DAY 6 — Checkerboard CA pattern

use sender culture: 5 mL receiver culture: 5 mL PBS: 10 mL optional fresh plate

do Build first: sender / receiver checkerboard Use same plate if signal stable, or a fresh one.

MINIMUM total consumables

bacteria/media LB broth used ≈ 140–160 mL plates used = 2–4 agar + 2 black plates reagents chloramphenicol stock used ≈ 150 µL AHL stock used = ~10–20 µL glycerol = 2 mL PBS = 30 mL

10c. What synthetic biology techniques did you utilize in validating this aspect of your final project? You can refer to the list of techniques in question 8. (min. 4 sentences)

11. Did you encounter any unexpected challenge(s) when performing your validation? If so, describe the challenge(s) and strategies to overcome it. If not, discuss potential problems, difficulties, limitations, and/or alternative strategies to overcome challenges in your final project. (min. 4 sentences)

SECTION 7: ADDITIONAL INFORMATION

12. List all references cited in this assignment (bullet-point list)

13. Create a supply list and budget for your project (bullet-point list)

• What supplies, equipment, and budget is needed for your project to work?

Requirement: DNA design / protein, wet lab (2 out of 4, use one from each category) Requirement: some data to show, data simulation Could be computationally heavy, could be lab heavy Aims 1,2,3 Computational Section (Kernel, Benchling) Lab Automation Section (Nebula, Opentrons, Nuclera) Wet Lab Assay/Testing

Group Final Project