Engineering Success | EHSBNU

Bacmid Design Strategy

We have constructed four specific bacmids (M, C, and S refer to containing MAHS, CAHS, and SAHS genes in the bacmids, respectively): the experimental design containing all three target proteins (MCS) and three negative controls (CM, MS, SC).

Expression System Design

Key Design Elements

Polyhedrin Promoter: A strong promoter commonly used in baculovirus systems for efficient expression

P2A Peptide Sequences: Enable ribosomal skipping during translation, ensuring production of individual proteins from a single transcript

Tandem Protein Arrangement: Three proteins arranged sequentially for coordinated expression

The three proteins were arranged in tandem and separated by P2A peptide sequences, which induced ribosomal skipping during translation and thereby ensured production of individual proteins from a single transcript.

Regulatory System Design

To further refine the system, we incorporated OsTIR1 as a regulatory element to facilitate protein degradation and promote cellular recovery after expression.

Auxin-Inducible Degradation System

To enable auxin-induced degradation, we fused an AID (Auxin-Inducible Degron) tag to the end of each protein sequence, positioned immediately upstream of the corresponding P2A site. This design allowed us to selectively degrade the expressed proteins by adding auxin after thawing, thereby accelerating cell recovery and improving viability.