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.
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:
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:
And so the proposed workflow pipeline would look like:
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.
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.
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.
The key challenge is host toxicity.
If the antimicrobial peptide is expressed directly, it may:
The proof-of-concept DNA fragment will encode a fusion protein containing:
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.
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.
A simplified construct layout is shown below: