Introduction

Synthetic biology constitutes an effort towards making biology easy to engineer [1]. This means that basic principles of engineering find their place in biological systems; We treat biomers as spare, interchangeable parts, the same way that we would approach the construction of any mechanical device. This necessitates the standardization of parts, in order to establish objectivity regarding their effectiveness and to promote the acceleration of knowledge [2,3]. For our project, to identify the optimal components and regulatory mechanisms for our system, we employed the Design-Build-Test-Learn cycle, allowing us to manipulate the expression of our constructs throughout different phases of the bacterial life cycle. To minimize cellular stress, we strategically divided our system into two phases: the exponential phase and the stationary phase. During the exponential phase, we expressed T7 polymerase and dsRNA molecules. In our pursuit of an autoinducible promoter active during the exponential phase, we explored the work of Zobel et al. and tested three of their synthetic promoters. Through our focused examination of their autoinducible properties, we determined that BG37 emerged as the optimal choice for regulating our system and we decided to further characterize BG37 by assessing its performance across various bacterial chassis, employing different plasmid backbones and carbon sources. By thoroughly characterizing this new basic part, we believe it will serve as a valuable tool for future teams aiming for orthogonal expression during the exponential phase.

Usage and Biology

The origin of BG37

Zobel et al. identified the BG synthetic promoters by systematically analyzing promoter activities in E. coli and Pseudomonas strains, particularly P. aeruginosa and P. putida. They found that the consensus sequences, especially the −10 and −35 regions, closely resembled those of sigma-70 promoters in E. coli, which suggested similar transcriptional mechanisms across these species. Using an initial plasmid-based selection in E. coli PIR2 cells, they efficiently screened for effective synthetic promoters, confirming their comparable activity in both E. coli and Pseudomonas [4].

We were particularly interested in using this specific promoter due to the assertion made in Huseyin Tas's PhD thesis that it is a "standard promoter that is more orthogonal, durable to environmental changes, and exhibits a correlated constitutive character throughout exponential growth across different media." This characteristic aligns well with our project's requirements for a reliable and versatile promoter [5].

Our characterization approach

During our experimental design, we aimed to answer key biological questions about how the BG37 promoter functions across different bacterial chassis, environmental conditions, and plasmid backbones. We sought to understand whether BG37 could maintain consistent, orthogonal activity during the exponential phase and how factors like growth media, carbon sources, and vector backbones influence its performance for optimal system regulation.

The characterization plan aimed to address several key biological questions, including:

1. Is BG37 an effective orthogonal promoter across different bacterial chassis? We aimed to collect more data on how BG37 functions in various bacterial strains to determine if it maintains consistent activity independent of host regulatory systems, ensuring its broad applicability and versatility [5].

2. How does BG37's activity compare to well-characterized promoters? This comparison aimed to establish BG37's relative strength and reliability, providing a benchmark against known standards. We conducted a thorough characterization by comparing BG37 with the standard Anderson J23119 promoter and the stationary osmy promoter, which allowed us to generate reliable and comparable data. This approach ensured that we could accurately assess BG37's performance in relation to well-established promoters. Characterization, being the process of estimating quantitative measures of part behavior, enabled us to quantify BG37's strength and activation’ s time, providing a solid foundation for its use in future applications.

3. How do environmental factors, such as carbon sources and growth media, impact BG37’s performance? Since various carbon sources lead to distinct metabolic products that can alter cellular physiology and energy availability, we aimed to assess if these metabolic shifts influence the promoter's activity. By testing BG37 with different carbon sources, we sought to determine whether changes in the cell's metabolic state would affect our system’s expression levels, ensuring its reliability in diverse growth environments [6].

4. Does BG37 function similarly across different plasmid backbone vectors? This question aimed to assess whether the promoter’s activity remains consistent across different vectors, which is crucial for its versatility in synthetic biology. Since various plasmid backbones are needed to work with different chassis, we compared the pSEVA23g19g1 vector with the pDGB3a1 vector, starting with E. coli DH5α as an intermediate step before transitioning to P. putida. By doing this, we gathered valuable data about the behavior of BG37 in different backbones, allowing us to understand how the promoter operates across diverse cloning vectors [5].

The general spirit for our characterization was standardization to the best of our ability. Given our need for an orthogonally sound promoter, we focused on the reproducibility of results, testing on different chassis, adequate controls to pinpoint phase activation and strength, and measurements that covered the entirety of the bacterial population’s life cycle. This lead to the gathering of detailed supporting data fit for a highly characterized Registry part. We hope that our efforts prove fruitful for future teams, as we add an autoinducible, synthetic, exponential phase- activated promoter.

Experiments

Experiments Our aim

Sequence and Features

Assembly Compatibility: