Design-Build-Test-Learn: Our Engineering Approach
Our project follows the Design-Build-Test-Learn (DBTL) engineering cycle, an iterative approach that allows us to continuously improve our system through multiple rounds of refinement. This methodology provides a structured framework for tackling complex problems in synthetic biology.
Our dry lab team focused on optimizing antimicrobial peptide production and testing systems. Through multiple engineering cycles, we developed efficient expression vectors, purification protocols, and activity assays.
Click on any node in the engineering cycle diagram to explore our iterative process in detail.
In the design phase, we utilized computational tools to identify and optimize promising antimicrobial peptide sequences. Key considerations included:
Our design process was informed by literature research, protein modeling, and consultations with experts in the field of antimicrobial peptides.
Improved peptide efficacy through strategic amino acid substitutions
Engineered vector with inducible promoter and fusion tags
During the build phase, we implemented our designs through molecular cloning and genetic engineering techniques:
We constructed multiple variants of our expression system to test different design hypotheses and optimization strategies.
Efficient one-pot cloning of multiple fragments
Optimized host selection for high-yield production
The testing phase involved rigorous characterization of our engineered systems:
Each construct was systematically evaluated to quantify performance and identify opportunities for improvement.
Antimicrobial activity against various bacterial strains
In the learning phase, we analyzed our results to extract key insights:
These learnings informed subsequent iterations of our engineering cycle, enabling us to refine our designs and experimental approaches.
Identified optimal induction timing and temperature
Discovered critical amino acid positions affecting function
We selected three promising antimicrobial peptide sequences from literature and designed expression vectors with a 6xHis tag for purification.
Synthesized gene fragments were cloned into pET28a vectors and transformed into BL21(DE3) cells for expression.
Expression was induced with IPTG and peptides were purified using Ni-NTA chromatography. Antimicrobial activity was tested against E. coli.
We observed low expression levels and peptide degradation. The direct fusion approach resulted in poor yield and activity.
Redesigned constructs with SUMO fusion partner to improve solubility and prevent degradation. Optimized codon usage for E. coli.
Cloned peptide sequences into pET-SUMO vector system. Incorporated TEV protease cleavage site for tag removal.
Tested expression under various conditions. Optimized purification protocol for SUMO-fusion proteins and TEV cleavage efficiency.
SUMO fusion significantly improved expression yield and solubility. TEV cleavage was efficient but required optimization of buffer conditions.
Based on our findings, we designed a library of peptide variants with systematic amino acid substitutions to enhance antimicrobial activity.
Generated peptide variant library using site-directed mutagenesis and PCR assembly. Validated constructs by sequencing.
Expressed and purified peptide variants. Conducted comprehensive antimicrobial activity screening against diverse pathogenic bacteria.
Identified key sequence determinants of antimicrobial activity. Discovered variants with enhanced potency against gram-negative bacteria.
Our human practices initiatives involved stakeholder engagement, educational outreach, and ethical considerations to ensure our project addresses real-world needs and concerns.
Click on any node in the engineering cycle diagram to explore our human practices work in detail.