Engineering Cycle Overview

Our engineering cycle focused on the synthesisation of barnacle cement's key structural protein Balcp19K (abbreviated to CP19K). CP19K plays a crucial role in underwater adhesion. The objective of this cycle was to create a plasmid sequence to be expressed within a bacterial host capable of CP19K synthesisation in the laboratory. This synthetic production enables testing of different proteases for cement protein degradation which can then be used in antifouling products such as biofilms, marine coatings or cleaning products.

Design Phase

Following the Design phase of the engineering cycle, two promoters (T7 and J23119) and two regulatory operators (LacO and TetO) were selected to form four promoter–operator combinations. These were chosen for their wide range of use. The goal at this stage was to identify which configuration could potentially achieve high protein yield.

Design Components Selected

  • Promoters: T7 and J23119
  • Operators: LacO and TetO
  • Combinations: Four promoter-operator configurations
  • Goal: Identify optimal configuration for high CP19K yield

Build Phase

The Build stage involved constructing a mechanistic model of CP19K expression under each promoter–operator combination using a system of coupled ordinary differential equations. These equations captured transcription, translation, and degradation processes, as well as repressor–inducer binding and ATP consumption, parameterised from literature data. Each system variant was simulated for 12 000 minutes in MATLAB to predict relative mRNA and protein output.

Mechanistic Model Components

  • Mathematical Framework: Coupled ordinary differential equations
  • Biological Processes: Transcription, translation, and degradation
  • Regulatory Elements: Repressor-inducer binding dynamics
  • Resource Management: ATP consumption modeling
  • Simulation Platform: MATLAB with 12,000 minute timeframe

Test Phase

During the Test and Learn phases, simulation results revealed that both T7-based systems outperformed J23119-based ones, producing approximately twice the amount of CP19K over the simulated period. This is matches with known biological characteristics of the 2 promoters and operators, as T7 is a strong bacteriophage promoter driven by T7 RNA polymerase, known for its high transcription rate. LacO marginally outperformed TetO by around 3 %, indicating slightly tighter repression control.

Key Test Results

  • T7 vs J23119: T7-based systems produced ~2x more CP19K
  • LacO vs TetO: LacO outperformed TetO by ~3%
  • Optimal Configuration: T7-LacO combination
  • Biological Validation: Results align with known promoter characteristics

Learn Phase

Overall, this engineering cycle demonstrated that the T7–LacO construct is most efficient for CP19K synthesis, also highlighting the effectiveness of computational modelling as a predictive and cost-efficient tool in synthetic biology. These results can then be used in the to test different proteases and identifying the most efficient CP19K-degrading enzyme.

Key Learnings

  • Optimal Design: T7-LacO construct identified as most efficient
  • Methodology Validation: Computational modeling proven effective
  • Cost Efficiency: Predictive modeling reduces experimental costs
  • Next Steps: Results inform protease testing strategy

Ni-NTA Purification Optimization

Design

To run the Ni-NTA and purify the protein after induced expression, we needed to calculate how much glycerol stock we should revive and how much broth to make. We calculated the optimum volume of broth to make, accounting for how much of our glycerol stock we were using and the total volume our centrifuge (Bioridge TGL-16.5M) could centrifuge at once. From these considerations, we decided 40ml of broth would be optimum to centrifuge.

Build

Following the protocol, we had discarded the supernatant and resuspended the cell pellets in lysis buffer. We then incubated, discarded the pellets and underwent the Ni-NTA purification process. We took 10ul aliquots every stage and added 5ul of 4x SDS-page loading buffer.

Test

Our SDS-page testing the protein purification revealed no bands. We suspected that the aliquots we had taken were too small and that the protein concentration was too little for visible bands to show up on the SDS-page

Learn

We revived a larger amount of glycerol stock, created 250ml of broth and took larger aliquots of 40ul.

Iteration Summary

  • Issue Identified: Insufficient protein concentration for detection
  • Root Cause: Small aliquot volumes and limited starting material
  • Solution Implemented: Increased broth volume and aliquot sizes
  • Scale-up: 40ml → 250ml broth, 10μl → 40μl aliquots