Our ambition is to develop a novel tool for synthetic biology which results in the CHOP system framework. Our construct was designed to be "customizable" in hopes to aid future iGEM teams and projects studying proteins compatible with our system.
Wide-range Application
Scale-up of the CHOP system is highly possible for industrial applications such as enzyme production. The demand for enzymes in industrial sectors is increasing rapidly due to their economical and ecological advantages. Amylase-one of the main industrial enzymes- production and research for the most optimized process is still ongoing (Aline et al 2018). The selection of the method to obtain enzymes should not only consider the rapidity and quantity but also optimized bioprocesses and cost-effective product recovery. CHOP system could produce enzymes with a simpler-friendly downstream process with high purity and great yield due to protein localization on the cell membrane or the vesicles. In addition, there is a need for more genetic studies with a focus on characterizing the enzyme that can integrate greatly under the CHOP system. Our CHOP sheds light on the progress made for the small steps towards these goals, starting with the discovery of new constructs and their corresponding genes.
The CHOP system is simple and the concept can be applicable to other systems: bacteria and yeast expression systems. The CHOP system with universal expression is expected to be a framework, in which we have tried to integrate the CHOP system with amilCP (BBa_K592009) and TEV protease (BBa_K1319004) as proteins of interest (PoI) expressed in E.coli. However, this CHOP system with universal expression does not rule out the possibility that it can be applied to other Gram-negative chassis. This is because the constituent components of this CHOP system, for instance, we used a strong promoter (BBa_J23100) that has been characterized in a wide range of hosts likes iGEM Keystone 2020 team applied it in Komagataeibacter rhaeticus that is an obligate aerobic Gram-negative. This wide range of hosts open the possibility to further application of the CHOP system in another recombinant protein in any host. Beside that, the evolved scaffold of the native OmpX, eCPX, expands the range of applications for bacterial display, especially in Gram-negative bacteria. Then we used artificial terminator (BBa_B1006) that calculated experimentally has 98% strength make it favor short rho-independent terminators with strong activity (i.e., with high free energy and long poly(U) tail) this benefit could be a necessary element for further in vivo implementation and expression (Rostain et al., 2015).
In pursuit of producing a blueprint for an ideal protein production system, our team encountered certain aspects in need of improvement. As a result, our team has provided insight to solve these setbacks in the form of molecular circuit designs in hopes to aid any future iGEM teams wishing to conduct further research on the topic.
Hypervesiculation
Studies have reported that several gene-deficient mutants relating to envelope stress and phospholipid accumulation in the outer leaflet of the outer membrane increase OMV production (Ojima et al., 2019). Previously, the UNSW_Australia team in 2016 successfully induced hypervesiculation of OMV in E.coli BL21(DE3) with various genetic alterations. We intended to use this mutant strain for developing the CHOP system to fulfil our goal of getting a higher yield. We made a T7 expression system version of the CHOP system that can be adapted to the mutant chassis.
Double terminal anchorage of Pols
After the CHOP system, we have plans to refine our circuit further by anchoring a PoI onto both of the extracellular terminals of eCPX, resulting in a biterminal display. eCPX scaffold permitted simultaneous display and labelling of distinct peptides on structurally adjacent C- and N-termini. Studies have also reported that the use of a long and unstructured linker can increase the accessibility of large proteins to peptides simultaneously displayed at both termini of eCPX, without substantially reducing the level of display (Rice & Daugherty, 2008)