Part:BBa_K3759000
mLCC
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 193
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage
Our group decide to enhance the activity of mLCC by proceeding two approaches, which are constructing a fusion protein of mLCC and hydrophobins and using the technique of Bacillus subtilis surface display.
The first approach is constructing the fusion protein which was made to enhance the efficiency of adsorption, since the surface of PET film is hydrophobic and the surface of mLCC is hydrophilic. By constructing the mLCC-linker-mHFBI, mLCC-linker-mHGFI and mLCC-linker-BsLA fusion protein, the PET degradation efficiency will be enhanced due to the unique properties of amphiphilicity and self-assembly of hydrophobins.
The second approach is using the technique of Bacillus subtilis surface display. By combining mLCC with the coat protein to form CotB-linker-mLCC, CotC-linker-mLCC, CotG-linker-mLCC and CotC-linker-mLCC fusion protein. mLCC will be immobilized on the Bacillus subtilis cell surface to obtain a recyclable whole-cell biocatalyst, which can reduce costs and make the mLCC more efficient degrading PET.
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.
Design Consideration
Create a fusion protein by jointing mLCC with hydrophobins：
The construct was cloned into a pET28a plasmid and transformed into BL21 (DE3) E. coli.
The construction includes:
1. a 6× His tag is added to enable us carrying out Ni-NTA protein purification
2. The CT fused with BslA, mHGFI or mHFBI with a GS linker (three Glycine Serine repeat: GGGGSGGGGS)
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. CotB is fused with mLCC at the NT with a GS linker (three Glycine Serine repeat: GGGGSGGGGS)
2. 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.
Protein Expression
Protein Expression
Figure 1. The expression of mLCC-linker-BslA (Left 1st 2nd), mLCC(Left 3rd 4th)，mLCC-linker-mHGFI(Left 5th 6th)，mLCC-linker-mHFBI(Left 7nd 8th)
Pre-expression:
The BL21 bacteria that contains aimed protein were cultured in 5mL LB liquid medium with kanamycin in 37℃ overnight. After taking samples, we transfer them into 1L LB medium with kanamycin.
Cultured in bottles:
After culturing in 37℃ in bottles, we used 0.5mM IPTG induced in 16℃ for 24 hours. Then, we used 200mM imidazole to eluting and get left 1st, 3rd, 5th, 7nd aimed protein, and we used 300mM imidazole to eluting the left 2nd, 4th, 6th, 8th aimed protein.
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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