Main Goal
The goal of our METALE system, represented by part BBa_K5422013, is to enable the controlled production of a modified Cyt2Aa1 toxin in the cytoplasm. Cyt2Aa1, a toxin naturally produced by Bacillus thuringiensis, is known for forming pores in the cell membranes of the intestinal barrier of insects like aphids, leading to their death. In our system, Cyt2Aa1 expression is driven by a strong ribosome binding site (RBS), ensuring high levels of toxin production.
At the same time, a weaker RBS regulates the production of T4 endolysin and T4 holin. When holin reaches a critical threshold, it induces pore formation in the bacterial membrane, allowing endolysin to penetrate and degrade peptidoglycan, ultimately leading to bacterial lysis and the release of large amounts of Cyt2Aa1. This results in aphid mortality. To fine-tune this mechanism, we plan to modify the RBS controlling the translation of T4 endolysin and T4 holin through site-directed mutagenesis. A library of 10 RBS variants will be generated to modulate their expression, using primers specifically designed to help select the most suitable mutant for our system.
Design - Proof of Concept
Before creating the final BBa_K5422013 part, we decided to build a proof of concept to validate our ideas using easily measurable genes. Our design involved linking various components together: a strong constitutive promoter, a strong RBS, GFP, another strong RBS, mCherry, and a terminator. While designing this sequence, we optimized codons for efficient expression in E. coli and ensured compatibility with BioBrick and Type IIS assembly standards. We also made adjustments to avoid secondary structures that could interfere with gene synthesis. These changes included synonymous codon substitutions to preserve the amino acid sequence while optimizing for synthesis and expression efficiency. Additionally, we designed the 10 sets of primers for performing RBS mutagenesis, which would be used based on fluorescence microscopy results. It was important to determine whether the length of the mRNA would affect translation levels, potentially making mutagenesis unnecessary.
Build
To construct our system, we assembled the different fragments using the Gibson Assembly kit from New England Biolabs. PCR-directed mutagenesis was then used to create the 10 RBS mutants. After transforming E. coli DH5α with our constructs, 8 of the 10 mutants were confirmed to be correct.
These 8 were purified and sent for sequencing to verify whether the mutagenesis was successful.
Test
Fluorescence microscopy showed strong expression levels of both GFP and mCherry before mutagenesis. Consequently, we proceeded with the mutagenesis experiments. The goal was to reduce mCherry expression relative to GFP by modifying the mCherry RBS.
However, fluorescence readings revealed consistently low levels of GFP and almost no mCherry expression across all strains, including the wild type. This indicates that the RBS modifications might have impacted mCherry mRNA stability or translation efficiency.
Even though a strong promoter was used, the near-zero levels of mCherry suggest issues with either the promoter or the translation initiation process. Interestingly, prior microscopy (see Results) had shown co-expression of both proteins, but spectrophotometric analysis failed to detect mCherry, pointing to discrepancies between the two experimental approaches. Further experiments, such as testing different RBS designs or bacterial strains, will be required to resolve these issues and optimize the system.
Learn
The spectrophotometric data were not usable and failed to validate the ideas proposed during the design phase. Nevertheless, fluorescence microscopy before mutagenesis clearly showed strong expression of both fluorescent proteins in the unmutated clone, indicating that the initial design was functional to some extent. These insights will guide the next iteration of the engineering cycle, particularly in refining the RBS design and addressing translation issues.