Team:SIS Korea/Parts
Testing out different parts was an essential procedure of improving our product to the fullest extent. In total, we created 64 parts, consisting of 19 basic parts and 45 composite parts.
The parts that allowed for the production of PETase were all more or less derived from BBa_K3288009, the wild-type gene derived from the original PET-degrading bacteria, Ideonella Sakaiensis. Some key parts of our testing involve: BBa_K3288055, our first composite part (also world first, at least to our knowledge) to utilize light-induced promotion in the production of PETase; BBa_K3288043, an improved constitutive expression part utilizing OXB20 instead of J23119 as the constitutive promoter which yielded stronger expression; BBa_K3288011, the first basic part to include NSP4 as the signal peptide displaying better secretion than the wild type; and BBa_K3288013 which is basically BBa_K3288011 but with 3 point mutations at the locations S121E, D186H, and R280A.
We improved our parts in many different ways. The primary improvement we made was by optimizing the codons of the wild type PETase sequence (BBa_K2110800) from Ideonella Sakaiensis to make it more suitable for E.Coli, resulting in the creation of BBa_K3288011. This was achieved by removing the endogenous signal peptide and replacing with the NSP4 signal peptide.
As mentioned before, we then further improved BBa_K3288011 into BBa_K3288013 by altering the PETase sequence of the previous part at S121E, D186H, and R280A. This increased enzyme activity and thermal stability, which allowed the E.Coli to endure further temperature regulation.
We improved our parts in many different ways. The primary improvement we made was by optimizing the codons of the wild type PETase sequence (BBa_K2110800) from Ideonella Sakaiensis to make it more suitable for E.Coli, resulting in the creation of BBa_K3288011. This was achieved by removing the endogenous signal peptide and replacing with the NSP4 signal peptide.
As mentioned before, we then further improved BBa_K3288011 into BBa_K3288013 by altering the PETase sequence of the previous part at S121E, D186H, and R280A increasing enzyme activity and thermal stability, which allowed it the E.Coli to endure further temperature regulation.Parts
Parts Intro/Overview:
