Part:BBa_K3759006
CotB-linker-mLCC-flag tag
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1374
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1009
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 463
Usage
Cell surface display is a technique to express the enzymes on the outer membrane of the carrier cells. This technique can exclude the process of protein purification which enormously reduced the cost, at the same time, easier to be reuse as the whole cell biocatalyst can be recycled by simple centrifugation.
In our design, we use Bacillus subtilis [1] as the carrier bacteria. mLCC has the optimal temperature at 70 degrees, which is not a durable temperature for most of the bacteria carriers. Bacillus Subtilis as the carrier cell has a short reproduction period and also has higher thermostability and adaptability toward basic environments [2]. Thus, we aim to surface display mLCC on Bacillus Subtilis to build a whole-cell catalyst.
Anchor protein is a surface-exposed coat component that has the purpose of displaying protein at the spore surface on the bacteria cells, function as an anchoring motif, support the attachment for the fused protein and chassis. We selected a well-studied anchor proteins by looking up references, namely CotB[4]. By constructing the CotB-linker-mLCC, the PET degradation efficiency will be enhanced.
Biology
Anchor protein is a surface-exposed coat component [3] that has the purpose of displaying protein at the spore surface on the bacteria cells, function as an anchoring motif, support the attachment for the fused protein and chassis. We selected a well-studied anchor proteins by looking up references, namely CotB[4].
The linker is GGGGSGGGGS.
LCC is a leaf-branch compost cutinase[5] and a kinetically robust protein[6]. A research published on Nature came up with a mutant enzyme, mLCC[1] that hydrolyzes 90% of PET in plastic bottles in just 10 hours. This is more efficient than any previous PET hydrolase, and more importantly, the resulting monomers- ethylene glycol and terephthalic acid have the same properties as the monomers found in petrochemical materials.
A flag-tag is added at the C-terminal to provide conditions for the use of fluorescence to detect the target protein after it is displayed on the cell surface.
Design Consideration
The construct was cloned into a pHT43 plasmid and transformed into B.subtilis BS168.
References
[1] Sang, Y. L., Choi, J. H., & Xu, Z. (2003). Microbial cell-surface display. Trends in Biotechnology, 21(1), 45-52.
[2] Cheon J , Kim S B , Park S W , et al. Characterization of L-Arabinose Isomerase in Bacillus subtilis, a GRAS Host, for the Production of Edible Tagatose[J]. Food Biotechnology, 2009, 23(1):8-16.
[3] Hosseini-Abari A , Emtiazi G , Lee S , et al. Biosynthesis of Silver Nanoparticles by Bacillus stratosphericus Spores and the Role of Dipicolinic Acid in This Process[J]. Applied Biochemistry & Biotechnology, 2014, 174(1):270-282.
[4] Xiao-Xia Y U , Tian J , Ning-Feng W U . Research Progress on Bacillus subtilis Spore Display
[5] Tournier, V. , Topham, C. M. , Gilles, A. , David, B. , & Marty, A. . (2020). An engineered pet depolymerase to break down and recycle plastic bottles. Nature, 580(7802), 216-219.
[6] Sulaiman S , You D J , Kanaya E , et al. Crystal Structure and Thermodynamic and Kinetic Stability of Metagenome-Derived LC-Cutinase[J]. Biochemistry, 2014, 53(11):1858-1869.
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