Contribution

Synthetic biology as a field often requires the design of complex genetic constructs composed of multiple consecutive transcriptional units. While such systems provide exceptional versatility of application and potentially enable the introduction of multi-step metabolic pathways into cells, they also entail certain fundamental limitations.

We would like to share our experience gained during the course of the project and draw attention to a key challenge: the repetitiveness of genetic parts used in constructing transcriptional units. Each functional transcriptional unit consists of elements such as a promoter, 5′ UTR, CDS, 3′ UTR, and poly(A) signal. While the CDS, representing the sequence of the gene of interest, typically exhibits high variability within a multi-unit construct, the other elements may recur with considerable frequency. This repetition carries significant practical implications. One major issue is the reduced pool of primers that can be designed to verify the presence of specific sequences. Primers located within repetitive regions often yield non-specific results, while primers designed for unique sequences may fail to ensure proper reaction performance in the absence of the targeted sequence. The limitation of primer availability also affects sequencing processes, which require a specific primer to initiate reading and nonspecific binding leads to mixed readouts. In constructs with highly repetitive genetic elements, the chosen cloning strategy itself may become a bottleneck. For example, Gibson assembly-based techniques can result in an increased prevalence of incorrectly recombined products.

A practical approach to overcoming these challenges is the use of diversified variants of promoters, UTRs, and terminators, sequences that differ at the nucleotide level yet retain comparable functionality e.g. rb glob and bGH poly(A) sequences from Asimov Mammalian Part Collection. When combined with standardized genetic part libraries, such as those offered by the Asimov collection, this strategy enables the construction of multi-element systems while maintaining high sequence specificity.

Another, yet extremely important in synthetic biology is the matter of safety and security. Hazard analysis is marked as one of critical steps in the design process of any synthetic biology product or project. Despite undeniable benefits coming from its inventions, the ability to engineer new forms of life may pose potential risks to societies and ecosystems, and is characterized by a high degree of uncertainty. Due to this fact the identification of threats and mitigation of risks is both a necessary and challenging activity.

We would like to share our experience gained during performance of System Theoretic Process Analysis (STPA) - an iterative hazard analysis tool developed by Massachusetts Institute of Technology professor Nancy Leveson. STPA is different from most techniques based on linear, chain-of-events accident causation models, which renders them ill-suited to be employed in the analysis of complex biological systems. However due to the uniqueness and novelty of the method, there are only a few sources that describe the technique. Therefore we prepared a simple guide as a complementary source for performing STPA by the future IGEM teams. It is broadened with examples more connected to the biological field and student life in hope that this will help with easier understanding of the method. Although STPA originally consists of three steps, we divided them into eight smaller steps to prevent overwhelm and confusion, due to the high complexity of the original three steps.

We hope that the guide will become a useful source of information for the IGEM community.