The Problem
In the well-known theory as central dogma, the genetic information stored in DNAs is first transcripted to RNA with the help of RNA polymerase and many other enzymes, and then RNA nucleotides are arranged into groups of three called codons to be translated into amino acids, which will eventually form different types of protein. These proteins and un-translated RNAs are the downstream gene expression product that serves as functional intracellular components or practical biological products. Yield improvements of these products are the fundamental topic of the whole biology field since it is of great importance for fields like biosynthesis, artificial genetic circuits, mechanism studies, and even their commercialization.
Taking the central dogma as a guiding theory, the conventional and straightforward means of increasing gene expression is either by increasing transcription or translation. In this sense, if we want to increase the downstream product, we have to adopt a stronger promoter so that more messenger RNAs will be transcripted from DNA, and adopt a stronger ribosome binding site so that more protein can be translated from mRNAs.
However, this increase in the inflow of mRNA and protein translation can increase cellular metabolic pressure, which may result in a lower growth rate and incompetency of the host cell. There are already numerous studies showing that higher expressions are accompanied by higher metabolic pressure most of the time, and this problem is even more serious for constructing genetic circuits with higher complexity.
Moreover, the degradation rate of RNA is quite significant in prokaryotes, with a lifetime of only 5 minutes in E. coli, increasing the need for more RNAs and stronger promoters or RBSs.
Our Solution
Our solution is degradation-tuning RNAs (dtRNAs). dtRNAs are hairpin-shaped synthesized structures that can be integrated into the 5' untranslated regions of mRNAs to influence their degradation rates. For conventional means like adopting stronger promoters, higher transcription rates inevitably lead to higher degradation rates, and much energy is put into the transcription process since degradations are fast in prokaryotic cells like E.coli. By decreasing the rate of mRNA degradation, significantly higher equilibrium concentrations of cellular mRNA can be achieved without secondary energy costs from the transcription process.
Therefore, unlike stronger promoters or RBSs, dtRNAs can increase gene expression without introducing an extra metabolic burden. It is demonstrated quantitatively by a mathematical model that describes the relationships of cellular growth rate and various kinetical parameters in gene expression.
Project Inspiration
We got the idea of dtRNA from a study conducted by Zhang and his team last year. dtRNAs are hairpin-shaped synthesized structures that can be added to the 5 prime untranslated regions of mRNAs to influence their rate of degradation. Unlike using a stronger promoter of RBS, dtRNAs can increase gene expression without introducing an extra metabolic burden. Adding different dtRNAs can also modulate the dynamics of gene circuits.
In Zhang’s study, they designed and tested a library of 82 synthetic dtRNAs and identified the functional structural features affecting RNA stability. These features include the loop size, stem length, and stem GC content as shown in this slide. Based on their findings, we selected six of the dtRNAs (dtRNA 1, 4, 19, 48, 68, 82), adjusted their parameters, and created new parts of our own.
In addition, their study could be extended by integrating these new parts into basic gene circuits such as toggle switches to further study the utilities of dtRNAs. We believe dtRNAs can serve as compact, ready-to-use biological parts for integration into existing genetic circuits and facilitate various downstream applications in the field of synthetic biology.
Human Practices
Before formally starting the project, we thought it is important to reach out to former iGEMers for advice, and thus we interviewed some of them to determine whether this project is truly helpful to the field of synthetic biology and whether there was any shortcoming in our plan. We also thought it is important to spread the influence of our project and iGEM competition as a whole so that more people, especially teenagers, might discover their interest in synthetic biology. Therefore, we carried out some educational activities in our local high school. To learn more, please go to the Human Practice page.