Team:PuiChing Macau/Design
Project Design
The overall goal of this project is to develop a new environmental friendly, cheap and easy-use flame retardant. We aim to achieve this through developing an engineered recombinant E.coli strain, given that this would allow a more easy and less polluted mass production. Moreover, we would also feed modified bacteria with different food waste, examining its efficiency in reaction.
1. Engineering Success
Previously, it has been proposed that biomaterial like protein would be a potential flame retardant resource. However, due to limited amount of research conducted for determining the best internal components (amino acid) of fire-resistant proteins, we thought to carry out the investigation to:
• Validate the feasibility of amino acid based flame retardant.
• Generate basic data for future protein flame retardancy ranking system.
• Establish prototype and procedure for flame retardancy testing in our school lab.
2. Flame Retardant Selection
• Since the bacteria E.coli is not specified for synthesizing flame retardant proteins, the essential gene would be cloned into E.coli to enable its synthesis. Based on the basic selection system for nitrogen-containing flame retardant protein proposed by Mingdao 2015 iGEM team, our team has developed a more comprehensive model, aiming to determine the highest Nitrogen % protein, hence the highest flame retardancy potential.
In consideration of the potential chemical toxicity from human practice, we have primarily focused our selection on the human and mammal proteins, and selected the SR protein that is identical to one of Mingdao 2015 iGEM team’s BioBricks.
• Food wastes are produced by a variety of sources, and extraction of high-value components, such as proteins from food wastes have been more accessible in recent years. The development in recycling waste components was due to their potential use in either nutritionally, pharmacologically, or as other functional ingredients.
For example, caesin protein was previously proposed to be a potential green phosphate-containing flame retardant, found in milk and cheese products. However, our team is without appropriate resources to extract enough caesin protein from expired products and food waste for flame retardancy testing. Therefore, we developed caesin producing recombinant E.coli strain using the BioBrick from 2019 Duesseldorf team's synthetic cheese and milk project.
3. Use of pET-11a as the Vector Backbone
In the pET system, the sequence of interest is transformed into E. Coli after being transcribed downstream from the T7 promoter and the gene 10 ribosome binding site. One can use Isopropyl β-D-1-thiogalactopyranoside (IPTG) induction of a chromosomally integrated cassette or infection with a polymerase-expressing bacteriophage lamba CE6 to achieve protein expression. In addition, protein expression would be temperately by lactose (Dubendorf and Studier, 1991; Hashemzadeh‐Bonehi et al., 2002). After induction to E. coli, the very active polymerase is expected to induce a high number of transcripts. Due to this phenomenon and the high efficiency of translation, the target protein may become the majority of cellular protein in only a matter of hours.
4. Addition of Adhesive Property
To reinforce its potential application in different aspects, the protein must be able to persist on different surfaces for a prolonged period of time. In approach, we fused the flame retardant protein with strong adhesive protein, such as mussel adhesive protein and cellulose-binding domain, targeting the creation of a more cost-effective product.
5. Future: Improve Downstream Processing Through Secretion System
Expectations were made where protein secretion could potentially reduce the downstream processing costs of its bioproduction, previously demonstrated by several iGEM teams using Type 1 Hemolysin secretion system. Of note, this secretion system is capable of the hemolytic toxin HlyA secretion. Given the N-terminal of HlyA functions as a signal peptide, engineered fusion protein with HlyA signal peptide and the target protein should enable the secretion process.
Reference:
1. Dubendorf J.W. and Studier F.W. (1991) Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. Journal of Molecular Biology. 2019(1) 45-59.
2. Hashemzadeh‐Bonehi et al. (2002) Importance of using lac rather than ara promoter vectors for modulating the levels of toxic gene products in Escherichia coli. Molecular Microbiology. 30 (3)