Part:BBa_K3759019

mLCC-linker-BsLA

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 261
Illegal EcoRI site found at 1079
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 261
Illegal EcoRI site found at 1079
Illegal NheI site found at 193
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 261
Illegal EcoRI site found at 1079
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 261
Illegal EcoRI site found at 1079
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 261
Illegal EcoRI site found at 1079
1000
COMPATIBLE WITH RFC[1000]

Usage

It has been well known that the surface of PET film is hydrophobic, and the surface of mLCC is hydrophilic. By constructing the mLCC-linker-BsLA fusion protein, the PET degradation efficiency will be enhanced enormously, due to the unique properties of amphiphilicity and self-assembly of hydrophobin BslA. Also, as BslA was extracted from bacteria and was a bacterial hydrophobin, it shows a better fusion with mLCC, which help the increment of the PET degradation efficiency of mLCC-linker-BslA.

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 GSGSGS.

BslA is a structurally defined bacterial hydrophobin that was found in the biofilm of Bacillus subtilis. It helps the assembling of TasA (an exopolysaccharide and an amyloid fiber-forming protein), the component of the biofilm matrix. BslA is composed of an Ig-type fold with the addition of an unusual, extremely hydrophobic “cap” region. The central hydrophobic residues of the cap are essential to allow a hydrophobic, nonwetting biofilm to form as they control the surface activity of the BslA protein. [3]

Design Consideration

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.

Protein Expression

Figure 1. The expression of mLCC-linker-BslA (Left 1st 2nd)

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 aimed protein, and we used 300mM imidazole to eluting the left 2nd 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.