Genetic Cubs Competing for Life

In nature, competition drives many crucial processes. Regardless of whether we consider a population of lion cubs or individual cells, it is the struggle of life that accelerates evolution. Recently, a research led by Dr Francesca Ceroni shed light on competition at the nanoscale, exploring how this effect takes place between genetic compounds in mammalian systems (Di Blasi et al., 2023). Let’s dive in and explore with them.?

What is resource competition??

The cell biochemically stays alive by expressing genes, producing compounds, and receiving energy from multiple reactions which is reinvested back into gene expression. The driving forces of energy generation and conservation are molecules, such as polymerases or ribosomes, that together form a resource stock for the cell to function. Like this, all cellular processes are coupled by their common resource stock. This leads to resource competition: for example, when two expression cassettes - regulatory sequences and target gene(s) in the vector DNA, meant to be expressed - need to be activated, they share the same pool of proteins that convene the reactions (Boo, Ellis & Stan, 2019). Naturally, resource competition might lead to smaller amounts and altered function of the (poly)peptides (a specific subgroup of molecules) produced. In #syntheticbiology, where the simultaneous introduction of multiple expression cassettes is of great interest, resource competition is one of the main bottlenecks for successful, high-rate and accurate bioproduction.?

As such, understanding the main molecular players controlling resource competition and having the potential to act as tunable elements reducing competition is crucial.??? #cells #bioengineering

So, what did the scientists find??

The researchers first introduced two competing plasmids, both producing fluorescent proteins, into the human kidney and Chinese hamster ovary cells. They investigated whether the increasing complexity of the expression cassette on one plasmid influenced resource competition between the two and the conclusion was affirmative. Interestingly, decreasing the number of transfected plasmids did not help with the competition.?

Then, they analysed the effect of the main design elements on the resource load. Three genetic parts in mammalian cells were explored: promoters, polyA tails, and Kozak sequences. These are DNA sequences initiating transcription, mRNA sequences terminating transcription and contributing to translation, and mRNA sequences initiating translation, respectively (Passmore & Coller, 2022). Tested individually and in combinatorial variation with each other, the three sequence types presented promoters as the most defining genetic element of the resource footprint of the genetic construct, with stronger promoters requiring more resources. Hence, the general conclusion stated that transcriptional resources might be more limiting than translational.

To investigate further the most optimal construct designs for synthetic biology #applications, the scientists tested the resource load of constructs with one or multiple transcriptional units. The best-performing constructs were those with smaller promoter numbers, which reinforced previous findings of limitations imposed by transcriptional resources.

To demonstrate the importance of these results, a TET-ON doxycycline-responsive system was tuned in design according to the previous work in which a single promoter initiated the production of two genetic compounds, rtTA and EGFP, as connected through an IRES or 2A peptide. This optimized cassette allowed for an improved fold change but did not yield higher production of EGFP. This directs scientists to future research, further optimisation, and a need to find a balance between the output range of interest and the suitable range of competition for resources. ????

Why is it important?

This #research showcases the #possibility of tuning genetic constructs to achieve lower resource competition in mammalian cells. At the same time, it provides future researchers with specific guidelines on how to make the co-expression systems more efficient. These guidelines serve as useful buoys for both fundamental and applied #biotechnology research. All to make genetic cubs less needy and more patient…

Author: Laima ?usta

Read here the article by Roberto Di Blasi et al. here: https://doi.org/10.1038/s41467-023-39252-4.

1.Boo, A., Ellis, T., Stan, G.-B. (2019) Host-aware synthetic biology. Current Opinion in Systems Biology. 14, p. 66-72. Accessed from: https://doi.org/10.1016/j.coisb.2019.03.001?

2. Di Blasi, R., Pisani, M., Tedeschi, F., Marbiah, M. M., Polizzi, K., Furini, S., Siciliano, V., Ceroni, F. (2023) Resource-aware construct design in mammalian cells. Nature Communications. 14. Accessed from: https://doi.org/10.1038/s41467-023-39252-4?

3. Passmore, L. A., Coller, J. (2022) Roles of mRNA poly(A) tails in regulation of eukaryotic gene expression. Nature Reviews Molecular Cell Biology. 23, p. 93-106. Accessed from: https://doi.org/10.1038/s41580-021-00417-y?