Wet Lab: System Implementation

Detailed protocols and characterization data for our engineered organism.

Project Overview

Our work focuses on engineering Pseudomonas putida KT2440 to express a novel enzyme pathway for plastic degradation. This pathway is contained within the pSEVA231 backbone.

Theoretical Note

P. putida was chosen for its robust metabolic capabilities and biosafety profile.

Gene Synthesis & Assembly

Gene Assembly Design

The gene cassette (BBa_KXXXXXXX) was synthesized with flanking BioBrick prefix/suffix sites.

Did you know?

The BioBrick standard was developed to make synthetic biology parts interchangeable and easier to assemble.

Assembly Protocol

Gibson Mix Preparation

# Prepare Master Mix (20 µL Total Volume)
Plasmid backbone (50 ng)      : 2 µL
Insert fragment (100 ng)      : 4 µL
Gibson Assembly Mix (2X)      : 10 µL
Nuclease-free water           : 4 µL

PCR Verification

Colony PCR was performed using VF2 and VR primers on selected colonies.

Bacterial Transformation

E. coli DH5a (Initial Cloning)

A heat shock protocol was performed on chemically competent cells.

Protocol Caution

Ensure cells remain on ice until the 42°C heat shock step to maintain cell viability.

P. putida KT2440 (Final Host)

Transformation of P. putida required electroporation due to its rigid cell wall.

Analysis & Characterization

Successful transformation was verified via Colony PCR and restriction digest.

Safety & Biosafety Notes

All work was conducted under Biosafety Level 1 (BSL-1) containment.

Successful transformation was verified via Colony PCR and restriction digest.