Since our project is the introduction of a new part in synthetic biology, it's quite versatile and flexible when it comes to practical implementation. We aim to introduce the compact and easy-to-use dtRNA to the whole iGEM community, so our direct end users are iGEMers and synthetic biologists. For background research, we have interviewed several experienced iGEMers and synthetic biology practitioners, together with the questionnaire from a wider perspective. The result shows that people from different research directions or teams on different tracks have a common appeal: how to reliably build complex, stable genetic circuits. Simple circuits are not enough for proper functions, but a complex one would face a serious problem with its practical behavior. The crux of the matter can be divided into two parts: the high-level metabolic pressure cause the instability of the chassis when the expression is high, or not enough signal changes to emerge against intrinsic noise in biological systems, and these two aspects are in mutual contradiction which can’t be solved simultaneously. In essence, this problem is a trade-off between gene expression and metabolic pressure, which has troubled synthetic biologists for many years. In this aspect, dtRNAs provide a perfect toolbox to solve this problem, as they could enhance the concentration of gene products without leading to a higher level of metabolic pressure.
Our experimental results show that the introduction of a single dtRNA structure could enhance protein expression up to nearly four-fold without any loss in growth speed, which solidified that degradation-tuning RNAs could control the level of downstream products in gene expression, having the potential to modulate dynamics of genetic circuits and regulate non-coding RNA functionalities by changing degradation rates. This not only benefits synthetic biologists by supporting the future construction of novel gene circuits with tunable parameters but can also benefit the entire field of biology. dtRNAs can help change the output behaviors of viral diagnostics, increase data accuracy of transcription quantifications, and even enhance expression in crude-extract-based cell lysates, which are substantially cheaper to produce but have higher RNase levels, reducing the cost for future biological experiments. The standardized biological parts form of dtRNAs would facilitate its integration into existing gene circuits and supports the future construction of novel biomolecular reaction networks with tunable parameters and increased complexity for other teams.
For implementation in the real world, we still need to consider:
1. The ease of molecular cloning: Since dtRNA is a new type of part that could be integrated in genetic circuits, the cost of molecular cloning would drastically increase if a convenient assembly scheme is not constructed. But since dtRNA are 10-60 bp-sized small fragments, it is compatible with most assembly methods that use overlapping primers containing dtRNA coding sequences and accessorial adaptor sequences as integration fragments in HiFi assembly, Golden Gate assembly, and Biobrick assembly. For practical consideration, even the RBS, promoter, and dtRNA could be synthesized as a whole part before assembling with other fragments.
2. The versatility of dtRNA across different host species. It is important when expanding this part to a wider field of study, many structural considerations that work in E.coli may be invalid, and another repeat of high-throughput screening may be necessary to select well-performed dtRNAs.
3. The fold change of yet first-generation dtRNAs is still waiting to be improved. This could be accomplished by iterative structural design combined with high-throughput screening, for a wider range of modulating degradation constants.
4. Safety considerations: The effect of horizontal transfer of dtRNA genes to wild bacterial specie or strains are yet unknown, further study could be carefully carried out before using this part outside of laboratories.