Team:BGU-Israel/Contribution
Our team added 10 new parts to the iGEM registry, for future use of iGEM teams. Among these parts, six are new basic parts and four are composite parts.
In addition, we made a useful contribution for future iGEM teams, by adding new functional information learned from literature to an existing Registry Part: BBa_K2139001.
We chose this biobrick as our favorite basic part since it is the core of our cellulolytic complex. As we have learned from a past iGEM team – the British Columbia 2016 team – about this cellulase, we hope that future iGEM teams would benefit from our work to improve its function and efficiency.
BBa_K3394000 consists of the coding region of Endo5A, an endo-1,4-beta-glucanase. Endo5a breaks down cellulose by cleaving the internal 1-4 beta glucose linkages in the cellulose chain.
The complete cellulose hydrolysis reaction involves this enzyme along with several other enzymes: endo-1,4-beta-glucanases, exo-1,4-beta-glucanases (or cellobiohydrolases) and beta-glucosidases. Endo5a is a relatively small protein with an optimal operating temperature of 50o C and an optimal pH range of 6.0-7.0. The NCBI GenBank number for Endo5a is HQ657203.1 (DNA sequence), AEB00655.1 (amino acid sequence).
We learned about the cellulase from a past iGEM team – the British Columbia 2016 team which fused it to the surface layer of Caulobacter crescentus (BBa_K2139001). In our project, we have performed codon optimization for its expression in Escherichia coli, and it was synthesized by IDT for us.
Endo5A was isolated from the bacterial flora found in the gut of a cotton bollworm (Helicoverpa armigera) which secretes a variety of plant-hydrolyzing enzymes.
Adlakha, N., Rajagopal, R., Kumar, S., Reddy, V. S., & Yazdani, S. S. (2011). Synthesis and characterization of chimeric proteins based on cellulase and xylanase from an insect gut bacterium. Applied and environmental microbiology, 77(14), 4859-4866.
Gupta, S., Adlakha, N., & Yazdani, S. S. (2013). Efficient extracellular secretion of an endoglucanase and a β-glucosidase in E. coli. Protein expression and purification, 88(1), 20-25.
We chose this biobrick as our favorite composite part because it is the main pillar of our project. This biobrick allows to display the cellulolytic complex on the surface of the genetically engineered bacteria and transforms it into a "Micro-Cellulose-Degrading-Machine". We hope that our work to improve its function and efficiency would benefit future iGEM teams.
Our project's goal is to decompose cellulose fibered wet wipes in the sewer systems to prevent the formation of "fatbergs" and clogs. Since cellulose cannot cross the cellular membrane, we engineered E. coli to express cellulolytic complex on its surface through AIDA-I auto-display system. For more information about AIDA-I auto-display system, see BBa_K3394006.
Our cellulolytic complex is composed of:
We have another design in which the order of the passenger genes is different (BBa_K3394008).
Jose, J., & Meyer, T. F. (2007). The Autodisplay Story, from Discovery to Biotechnical and Biomedical Applications. Microbiology and Molecular Biology Reviews, 71(4), 600–619.
Amir, L., Carnally, S. A., Rayo, J., Rosenne, S., Melamed Yerushalmi, S., Schlesinger, O., ... & Alfonta, L. (2013). Surface display of a redox enzyme and its site-specific wiring to gold electrodes. Journal of the American Chemical Society, 135(1), 70-73.
In our project we built a cellulose degrading complex – a "Micro-Cellulose-Degrading-Machine". Some parts of the complex were discovered by previous iGEM teams but had a different DNA sequence (for example: optimized for a different chassis). Thus, we added some similar basic parts which are somewhat different in sequence and function.
Our collection offers the cellulose degrading complex as a whole and as separated individual parts.
Parts overview
We are proud to present the parts we designed for our project!
Part's link
Type
Part's name
Short description
BBa_K3394000
Basic
Endo5a Cellulase
Coding Sequence of Endo5a cellulase (for E. coli)
BBa_K3394001
Basic
CBD
Coding Sequence of CBD (Cellulose Binding Domain)
BBa_K3394002
Basic
Histidine tag
Coding Sequence of 6-histidine tag (His-tag)
BBa_K3394003
Basic
Glycine-serine (GS) linker
Coding Sequence of Glycine-serine (GS) linker
BBa_K3394006
Basic
AIDA-I auto-display system
Coding Sequence of AIDA-I auto-display system
BBa_K3394009
Basic
ICE
Coding Sequence of ICE (Integrative and Conjugative Element)
BBa_K3394004
Composite
Cellulolytic complex [1]: His-Endo5a-GS-CBD
Coding Sequence of the Cellulolytic complex, cellulase Endo5a at the N-terminus
BBa_K3394005
Composite
Cellulolytic complex [2]: His-CBD -GS- Endo5a
Coding Sequence of the Cellulolytic complex, CBD at the N-terminus
BBa_K3394007
Composite
Cellulolytic complex [1] with AIDA-I
Cellulolytic complex [1] with AIDA-I auto-display system
BBa_K3394008
Composite
Cellulolytic complex [2] with AIDA-I
Cellulolytic complex [2] with AIDA-I auto-display system
Favorite Basic Part: BBa_K3394000
Figure 1: Cellulose chain. 1-4 beta glucose linkage is highlighted
References
Favorite Composite Part: BBa_K3394007
Figure 1: Illustration of decomposition of cellulose fibered wet wipes by engineered E. coli.
Figure 2: Complete cellulolytic complex expressed on the bacteria's surface by AIDA-I auto-display system
References
Collection
Part's link
Part's name and description
BBa_K3394000
Endo5a Cellulase
BBa_K3394001
CBD (Cellulose Binding Domain)
BBa_K3394002
Histidine tag
BBa_K3394003
Glycine-serine (GS) linker
BBa_K3394006
AIDA-I auto-display system
BBa_K3394004
Cellulolytic complex [1]: His-Endo5a-GS-CBD
BBa_K3394005
Cellulolytic complex [2]: His-CBD -GS- Endo5a
BBa_K3394007
Cellulolytic complex [1] with AIDA-I
BBa_K3394008
Cellulolytic complex [2] with AIDA-I