Improving PET biodegradation

The 2021 Edinburgh iGEM Team designed parts for PET biodegradation using various silica tags, and we built upon that in our project. The 2021 Team used the Dou-PETase (BBa_K3946023) double mutant for their designs. We tested two other PETase mutants, Tri-PETase (BBa_K4390074) and FAST-PETase (BBa_K4390073), and we found that FAST-PETase has improved PET degradation ability. Hence, the new FAST-PETase part is an improvement over Dou-PETase

Improving the biosensor

Our biosensor construct was based off the 2020 Edinburgh iGEM Teams design, who produced a Transcription only (TXO) ArsR regulated biosensor, under T7 promoter expressing iSpinach (BBa_K3380600), for the detection of arsenic in solution. We improved this in three ways:

1. The Edinburgh 2020 team used the iSpinach aptamer for its biosensor construct. However, iSpinach has lower fluorescence than Broccoli [1], so we designed a similar construct using Broccoli (BBa_K4390105)
2. The Edinburgh 2020 team used the ArsR transcription factor in their cell free system for detecting arsenic ions. We expanded the repertoire of transcription factors by including MerR (BBa_K4390097) and PbrR (BBa_K43900100), which bind to Hg²⁺ and Pb²⁺ respectively, making it possible to test samples for contamination with mercury, lead or arsenic.
3. We used a different T7 promoter without an internal BsaI site, allowing for Type IIS assembly of this biosensor using JUMP. The new part is found under BBa_K4390103.

References

[1] Millacura FA, Li M, Valenzuela-Ortega MA, French C. TXO: Transcription-Only genetic circuits as a novel cell-free approach for Synthetic Biology. bioRxiv. 2019.