best kozak sequence for mammalian expression

As the test plasmid uses more resources, less are available for the expression of the capacity monitor, leading to a decrease in its expression levels. When co-expressed in the cell, the monitor and the test plasmid rely on the same pool of cellular resources for gene expression. To visualise and quantify intracellular resource usage, a test plasmid is co-transfected along with a fluorescence-based capacity monitor, a CMVp-controlled cassette for mKATE expression 15. To advance resource-aware construct design in mammalian cells, we developed a framework for quantification of the load imposed by different designs on cellular resources. Understanding resource competition and its impact on construct performance is particularly important when co-expression of different cassettes is desired, something that is often sought after when engineering mammalian systems, from the use of transfection controls in fundamental research to the expression of multi-gene constructs for bioproduction and biotherapeutic applications 17, 20, 21.Ī framework to quantify the resource load of genetic parts

However, accurate characterisation of the demand that different construct components place on cellular resources is still missing in mammalian cells, limiting our ability to rationally design genetic constructs with minimised resource footprint towards robust and predictable engineering of these organisms. Evidence confirmed that gene expression resources are as limiting in mammalian cells as they are in bacteria and that unobvious coupling of genetic circuits can arise due to synthetic cassettes drawing from the same pool of resources 15, 16, 17, 18, 19. Among these, identification of genetic designs with lower impact on the host resources was shown to be feasible and desirable for optimising synthetic construct performance 5, 14.Īlthough robust heterologous expression is central to mammalian cell engineering and its applications, resource competition in eukaryotes has only recently started to be investigated.

In bacteria, the phenomenon has been extensively assessed and quantified 4, 5, 6, and many experimental and computational strategies towards its mitigation have been proposed 7, 8, 9, 10, 11, 12, 13. polymerases, ribosomes, and other cellular factors) by different expression cassettes, leading to a non-intuitive indirect coupling of their output levels and behaviour 1, 2, 3. Resource competition arises from the simultaneous use of cellular gene expression resources (i.e.

Best kozak sequence for mammalian expression