Part:BBa_K5487003

Name: Enhanced Surface Display of Z1-PETase and MHETase Fusion Protein

Function: This composite part is designed for the regulated and efficient surface display of the Z1-PETase and MHETase fusion protein on bacterial cells, utilizing the Lpp-OmpA system. The construct is intended for applications in biodegradation of plastics, biocatalysis, and potentially as a platform for other environmental or industrial processes.

Components and Mechanism:

T7 Promoter: A strong and tightly controlled promoter that initiates the transcription of the downstream genes. It is inducible by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) or through interaction with the T7 RNA polymerase.

lac operator: A regulatory sequence that, in conjunction with the lac repressor, allows for the tunable repression of the T7 promoter in the absence of IPTG, ensuring that the expression of the fusion protein is tightly regulated.

RBS (Ribosome Binding Site): A sequence that precedes the coding region of the fusion protein, facilitating the binding of ribosomes and initiating translation.

Lpp-OmpA: A fusion protein construct that anchors the Z1-PETase and MHETase to the bacterial outer membrane, with Lpp acting as a signal peptide and OmpA as a stable membrane anchor.

Z1-PETase: An engineered variant of the PET hydrolase from Ideonella sakaiensis, which is capable of degrading polyethylene terephthalate (PET).

MHETase: An enzyme responsible for the hydrolysis of monomeric units of PET, such as mono(2-ethylhexyl) terephthalate (MHET).

Optimal Conditions:

Temperature: Typically around 37°C for bacterial growth and protein expression. Media: An appropriate bacterial growth medium, such as Luria-Bertani (LB) broth, supplemented with the necessary antibiotics and inducers.

Application:

Biodegradation of PET: The surface-displayed Z1-PETase and MHETase can work synergistically to break down PET plastics into their monomers. Biocatalysis: The fusion protein can be used for the biocatalytic conversion of PET and related compounds in industrial processes. Environmental Remediation: This construct could be used in bioremediation strategies targeting plastic pollution.

Usage in Projects: To use this composite part in a project:

Transformation: Transform the construct into a suitable bacterial host, such as E. coli. Induction: Induce the expression of the fusion protein by adding IPTG to the culture or by using a T7 RNA polymerase system. Verification: Confirm the surface display of the fusion protein using techniques such as immunofluorescence or flow cytometry. Application: Utilize the bacterial cells for biodegradation assays, biocatalysis, or other relevant applications.

Safety Considerations:

Handle bacterial cultures and recombinant DNA with appropriate biosafety precautions. Use personal protective equipment (PPE) and follow good microbiological practices.

Storage and Stability:

Store bacterial glycerol stocks at -80°C for long-term preservation. Maintain cell cultures under stable conditions to prevent loss of surface-displayed protein functionality.