Part:BBa_K3759021
linker-mLCC-flag tag
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 297
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 297
Illegal NheI site found at 229 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 297
- 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 297
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 297
- 1000COMPATIBLE WITH RFC[1000]
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, CotC, CotG, COtX[4]. By constructing the CotB-linker-mLCC, CotC-linker-mLCC, CotG-linker-mLCC, CotX-linker-mLCC the PET degradation efficiency will be enhanced.
Biology
LCC is a leaf-branch compost cutinase[1] and a kinetically robust protein[2]. 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.
The linker is GGGGSGGGGS.
Design Consideration
Construct Cell surface display of mLCC in Bacillus subtilis：
The construct was cloned into a pHT43 plasmid and transformed into B.subtilis BS168 The construction includes:
1. 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.
References
[1] 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.
[2] 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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