Team:Hong Kong CityU/Contribution
Contributions
Our team is delighted to contribute to the iGEM community
and the future iGEM teams with the following items:
1. The modification of the T7 promoter
The T7 promoter is a biobrick part that has been widely used by a number of iGEM projects in previous years. This year, we have proposed the use of a new T7 promoter (C62-T7 mutant promoter; BBa_K3511008). The C62-T7 mutant promoter is reported to facilitate enhanced in vitro transcriptional activity by more than 10-fold relative to the wild-type T7 promoter (Paul et al., 2013). This new biobrick adds to the existing collection of T7 promoter variants and will be available to the iGEM community. It will provide an additional choice for future iGEM teams needing a strong promoter to enhance gene expression.
2. New add-in items and modification of the plastic
degradation biobricks in the iGEM part registry
Biodegradation of plastic is a popular topic in the iGEM competition every year. Our team has created two new parts this year: the mutant papain-G23W (BBa_K3511000) and mutant PueA-R392F (BBa_K3511001) enzyme biobricks. These two mutant biobricks are derived by in silico mutagenesis of two wild-type polyurethane (PU)-degrading enzyme genes, papain and polyurethanase esterase A (PueA) (Bhardwaj et al., 2012). In silico analysis indicated that the two mutant enzyme proteins possess a higher binding affinity for PU plastic and therefore likely to catalyze more efficient breakdown of PU. These biobricks will serve as an additional resource for future iGEM teams working on a similar project theme, and could possibly help in their project design workplan..
In addition, our team has used the polyethylene terephthalate hydrolase (PETase) biobrick (BBa_K2013002) previously designed by the iGEM16_UESTC-China team to construct the composite biobrick, BBa_K3511002 which consists of Papain-G23W (BBa_K3511000), PueA-R392F (BBa_K3511001), and PETase (BBa_K2013002), and are linked together with a GSG linker (BBa_K3511002). BBa_K3511002 represents the core composite part of our project which supports the coexpression of the three plastic-degrading enzymes, PETase, papain-G23W, and PueA-R392F. The biobricks designed in this project can serve as useful tools for future iGEM teams aiming to develop novel solutions to tackle the plastic pollution problem.
New parts were added in the part’s Registry page with the following information. For example, according to the research article by Paul et al. (2013), the mutant C62-T7 promoter is reported to enhance in vitro transcriptional activity of an expression plasmid by more than 10-fold relative to the wild-type T7 promoter.
The papain and PueA enzymes have been reported to have the ability to degrade polyurethane plastic. Our team has introduced a single-base change to the genes encoding these 2 enzymes, and in silico analysis indicated an increase in binding affinity of both mutant enzymes for PU plastic. For the mutant G23W papain, we have changed the glycine residue to tryptophan at the 23rd position. For the mutant PueA-R392F enzyme, we have changed the arginine residue to phenylalanine at the 392 position.