The diffusion of reaction intermediates limits the efficiency of many industrial biocatalytic pathways. The UNSW iGEM team has designed the Assemblase self-assembling scaffold system as the solution to this problem.

The Assemblase scaffold specifically and covalently co-localises enzymes in a modular system. As a result, substrate can be channelled between enzymes at a much more efficient rate, thanks to the increased concentration of metabolic intermediates in the immediate surroundings of the enzymes. This may increase the rate of multi-step enzymatic reactions.

Modularity is desirable as it means the scaffold can be easily adapted for use in a range of pathways important in industry, bioremediation, and pharmaceutical synthesis. Proof of principle was established using enzymes from the indole-acetic-acid biosynthesis pathway.

The scaffold is a heterohexamer of two alpha prefoldin and four beta prefoldin subunits, each of which has been fused to either a Spy or Snoop Catcher. These catchers covalently bind to Spy or Snoop Tags fused to enzymes of interest.

Advantages of the scaffold include that prefoldin is highly thermostable and chemically resistant, being a protein derived from thermophilic archaea. This permits our scaffold to be used at high temperatures, allowing for increased kinetic energy in our system and therefore an increased rate of catalysis.

The use of Spy and Snoop Tag/Catchers gives our scaffold modularity with low cross-reactivity, because of the specificity of the irreversible bonds. The tags are also small, so are unlikely to interfere with enzyme functionality when fused to them.

Over the past few months we have been busy cloning DNA, expressing and purifying some really cool proteins, and attaching proteins together through self-assembly and with the Spy/Snoop Catcher/Tag system. We also got to perform some really awesome enzyme assays and FRET experiments, alongside modelling our enzyme kinetics with some mathematical magic. We also performed molecular dynamics analysis, and grew our very own plants on agar plates in the lab! We had a great year and would absolutely love to share it with you. So, have a look around, and explore all things UNSW iGEM!