Part:BBa_K1582002
inJanus-m
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 121
Usage
Janus is a kind of amphipathic protein which could self-assembly spontaneously. Due to its special properties, we could make many new applications. We use them as substrate to fix antibodies on a high-flux tumor detection chip. Meanwhile, they are used to catch cutinases for plastic degradation. We even make them into a fusion to test if the enhancement could be better. And we use its amphipathicity to achieve protein separation, where they act as a special purification tag, and the system could be as simple as polymer, detergent and water.
Biology
Janus could be produced by filamentous fungi, such as Ascomycetes and Basidiomycetes, and their scientific name is hydrophobin. Many different aspects of fungal development have been attributed to Janus. For example, they are thought to play a role in the formation of aerial hyphae and fruiting bodies. One of the most important features of Janus is that they are able to assemble spontaneously into amphipathic monolayers at hydrophobic–hydrophilic interfaces.
There are two classes of Janus, which are divided by the stability of their self-assembly. inJanus from Grifola frondosa belongs to Class I. They could generate very insoluble assemblies, which can only be dissolved in strong acids such as trifluoroacetic acid or formic acid. We did some mutations to it, in order to make it expressed in E.coli, and we call it inJanus-m.
Protein Expression
Pre-expression:
The bacteria 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 4 hours culturing in 37℃ in bottles, we used 500μM IPTG induced in 16℃ for 8-12h. We used McAc 0 and 20 to wash off the bulk proteins, and used McAc 200 to wash off aimed proteins.
Figure 1.The expression of inJanus-m
We finally can use McAc 0 and 20 to wash off the bulk proteins and use McAc 200 to wash off aimed proteins.
Improvements
Group: BJEA_China 2021
Author: Kairuo Zhang
Summary:Hydrophobins are small surface-active proteins, and have both fungal and bacterial origins. Hydrophobins originated from fungi are divided in to two class, and are being widely used and applied. HGFI[1] is hydrophobin derived from fungi, and HGFI is a class I hydrophobin. Hydrophobin’ s hydrophobic part is exposed on the surface forming a planar area called the hydrophobic patch, making the surface of the protein contain both hydrophobic and hydrophilic area therefore making the surface making the hydrophobins amphiphilic. Because the hydrophobins’ amphiphilicity property it can self-assemble themselves with others. In 2015, the team Tianjin mutated this hydrophobin, and the resulting mHGFI (inJanus-m, BBa_K1582002 ) can be expressed in bacteria.Recently, a research published on Nature came up with a mutant enzyme, mLCC[2]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.
We construct 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-mHGFI：BBa_K3759018 fusion protein, the PET degradation efficiency will be enhanced due to the unique properties of amphiphilicity and self-assembly of hydrophobins.
Protein Expression
Figure 1. The expression of mLCC(Left 3rd 4th)，mLCC-linker-mHGFI(Left 5th 6th)
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 3rd, 5th aimed protein, and we used 300mM imidazole to eluting the left 4th, 6th aimed protein.
References
[1] Song D , Wang X , Gao Z , et al. Expression, Purification and Characterization of Hydrophobin HGFI from Grifola Frondosa in Saccharomyces Cerevisiae[J]. Acta Scientiarum Naturalium Universitatis Nankaiensis, 2018.
[2] 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.
| None |