Individual Final Project
Final Project Idea: Opentrons-Integrated Modular Peptide Secretion Platform
Overview
My final project explores the development of a modular peptide production platform that combines microbial expression, secretion engineering, and low-cost automation. The aim is not simply to produce one peptide, but to build a reusable workflow that can be adapted for many different peptide products.
The core idea is to design a plug-and-play system in which a peptide-encoding DNA sequence can be inserted into a standardised plasmid architecture containing the regulatory and functional components needed for expression, secretion, and downstream processing. This construct would then be introduced into a microbial host for production.
To strengthen the workflow, I want to pair this platform with Opentrons-based automation for demonstration purposes at LifeFabs and also with the ambition of incorporating a newly designed Gel Electrophoresis module for the opentrons to be able to use efficiently as i will explain in the proposed Opentrons based pipeline below. This would allow parts of the DNA assembly and screening process to be standardised and scaled more efficiently, creating a semi-automated pipeline from construct preparation to production testing.
Why this project matters
Short pASeptides are increasingly important in biotechnology, therapeutics, and research. However, peptide production can often be expensive, difficult to scale, and purification-heavy. Instead of approaching peptide manufacturing as a one-off design problem, this project focuses on creating a modular production platform that could be reused across different peptide targets.
The broader motivation is to make peptide production more:
- accessible
- reproducible
- scalable
- automation-friendly
Core project concept
The platform is based on the idea of building a standard DNA construct for peptide production. A peptide-coding insert would be placed into a plasmid containing features such as:
- promoter and ribosome binding site for expression
- fusion or stabilisation tag
- peptide payload
- protease cleavage site
- terminator
- secretion-related elements
- optional affinity tag for purification
And so the proposed workflow pipeline would look like:
- Select the host organisms gene coding for the peptide of interest and take a DNA sample
- After research of the peptides gene sequence find a corresponding enzyme digest to cut the desired DNA sequence out
- Run this on a gel electrophoresis board prototype designed to work within opentrons
- Program opentrons to then identify and pull up the corresponding DNA band coding for the gene of interest
- Run a purification procedure
- Then Do a bacterial transformation procedure to insert this into our “DNA Cassette” i.e. E. coli for its many benefits
- Allow it to divide and produce the desired peptide
- Purify the peptide fluid
- (Optional) run a purity test to see how well the peptide was synthesised
Demonstration Idea: Using Antiomicrobial peptide synthesis:
Context: Antimicrobial peptides (AMPs) are small, naturally occurring defense molecules produced by nearly all organisms as a rapid first line of innate immunity against bacteria, fungi, viruses, and cancer cells. As potent alternatives to conventional antibiotics, they act by disrupting microbial membranes or modulating immune responses, holding promise for treating multidrug-resistant infections.
“As an initial proof-of-concept, the platform will be validated using GFP in E. coli to demonstrate construct assembly, expression, and secretion behavior before extending the system to functional peptide payloads.”
For the initial proof of concept, the project focuses on expressing a small antimicrobial peptide in E. coli using a fusion-based design that reduces toxicity during production.
Problem
Antimicrobial peptides are powerful molecules that can inhibit or kill bacteria. They are interesting alternatives to traditional antibiotics because many act through membrane disruption or broad physical mechanisms rather than single enzyme targets.
However, producing antimicrobial peptides inside microbial hosts creates a major challenge:
The host cell may be damaged or killed by the same antimicrobial peptide it is producing.
This is especially relevant when using E. coli as the production organism. Although E. coli is widely used for recombinant protein expression, it may be sensitive to antimicrobial peptides if they are produced in an active form inside the cell.
Therefore, a simple peptide production system needs a way to keep the peptide inactive, protected, or masked during expression, while still allowing recovery of the active peptide later.
Updated Proof-of-Concept Choice
The original project idea considered using nisin as the target peptide. However, nisin is relatively complex for a first proof-of-concept because it is a lanthipeptide that requires post-translational modifications for full biological activity.
For a simpler first demonstration, the project will instead use a cecropin-style antimicrobial peptide as the model target.
Cecropins are small antimicrobial peptides originally discovered in insects. They are useful as a proof-of-concept target because they are short, peptide-based, and directly linked to antimicrobial activity.
The project will not initially attempt to build a fully optimised therapeutic production system. Instead, the goal is to demonstrate the principle that a modular genetic cassette can be designed to express a peptide payload in a controlled and recoverable format.
Core Design Challenge
The key challenge is host toxicity.
If the antimicrobial peptide is expressed directly, it may:
- damage the E. coli membrane
- reduce host growth
- decrease expression yield
- select for mutations that silence or reduce peptide production
- make the system unreliable as a production platform
To address this, the peptide will be expressed as part of a fusion construct rather than as a free active antimicrobial peptide. (edit: I realise that cell-free systems would solve this too, but it would diminish the pipeline of the projects initiative.)
Proposed Fusion Construct
The proof-of-concept DNA fragment will encode a fusion protein containing:
- His-tag
The His-tag provides a simple purification handle. It allows the fusion protein to be purified using affinity-based purification methods. In this project, the His-tag is included to make the downstream recovery process more standardised and compatible with a modular platform.
- SUMO tag
The SUMO tag acts as a solubility and shielding partner. It can help the host cell produce the peptide payload in a less toxic, more stable fusion form.
For this project, the SUMO tag has two main purposes:
improve expression and folding of the fusion product reduce the chance that the antimicrobial peptide is active during production
This makes the peptide easier to produce in E. coli without immediately harming the host.
- Cecropin peptide payload
A simplified construct layout is shown below: