Part:BBa_K3759000

mLCC

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 193
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE 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.

Molecular Cloning

Create a fusion protein by jointing mLCC with hydrophobins

For Molecular cloning, we selected pET28a as vector. We successfully amplified four gene segments of mLCC (as control group), mLCC-linker-mHFBI, mLCC-linker-mHGFI and mLCC-linker-BslA. Then we digested and connected the four segments to pET28a vector through two restriction enzymes of BamHI and XhoI. At present, four recombinant plasmids have been successfully constructed.

Figure 1. (a). M: marker; Lane 1: mLCC 780bp; Lane 2: mLCC-linker-mHGFI 1059bp Lane 3: mLCC-linker-mHFBI 1035bp; Lane 4: mLCC-linker-BslA 1227bp
(b). M: marker; Lane 1,2,3,4: mLCC 780bp; Lane 5,6,7: mLCC-linker-mHGFI 1059bp Lane 8,9.10: mLCC-linker-mHFBI 1035bp; Lane 11,12,13: mLCC-linker-BslA 1227bp )

Cell surface display of mLCC in B-subtilis

During cloning we used pHT43 plasmid. We successfully performed PCR amplification of all the four fusion genes. As we continued our lab, recombinant plasmid with CotB was successfully constructed after the double digestion verification (pHT43-cotB-linker-mLCC). However, the other three CotC, CotG, and CotX are still continuing to be tried out.

Fig. 2. (a). M: marker; Lane 1: cotB-linker-mLCC; Lane 2: cotC-linker-mLCC; Lane 3: cotG-linker-mLCC 1426bp Lane 4: cotX-linker-mLCC 1357bp
(b). M: marker; Lane 1: cotB-linker-mLCC 1986bp ; Lane 2: pHT43-cotB-linker-mLCC (after construction);
(c). cotB-linker-mLCC transformed into pHT43 plasmid

Protein Expression

Create a fusion protein by jointing mLCC with hydrophobins

We transformed four recombinant plasmids into BL21 expressing strains. For four recombinant strains, we tried three IPTG induction concentrations of 0.5mM, 0.7mM, 1.0mM and three induction times of 16h, 20h and 24h, respectively. We found that the induction concentration of 0.5mM IPTG and the induction time of 20h were the best.

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.