Abstract
The presence of standardised tools and methods to measure and represent accurately biological parts and functions is a prerequisite for successful metabolic engineering and crucial to understand and predict the behaviour of synthetic genetic circuits. Many synthetic gene networks are based on transcriptional circuits, thus information on transcriptional and translational activity is important for understanding and fine-tuning the synthetic function. To this end, we have developed a toolkit to analyse systematically the transcriptional and translational activity of a specific synthetic part in vivo. It is based on the plasmid pTRA and allows the assignment of specific transcriptional and translational outputs to the gene(s) of interest (GOI) and to compare different genetic setups. By this, the optimal combination of transcriptional strength and translational activity can be identified. The design is tested in a case study using the gene encoding the fluorescent mCherry protein as GOI. We show the intracellular dynamics of mRNA and protein formation and discuss the potential and shortcomings of the pTRA plasmid.
Highlights
In the field of Synthetic Biology and bioengineering genetic parts with specific biological functions are manipulated or introduced into a host cellular system
Already good progress is made in the development of standards for genetic parts, as represented by the library of BioBricks and biological parts with its extension by the iGEM students’ competition, and the Standard European Vector Architecture (SEVA; http://seva.cnb.csic.es), a repository of formatted molecular tools [3,4]
The centrepiece of the method is plasmid pTRA, which was designed for dynamic analysis of heterologous gene expression (Fig 1A)
Summary
In the field of Synthetic Biology and bioengineering genetic parts with specific biological functions are manipulated or introduced into a host cellular system. For the successful implementation of such functions it is crucial to understand and predict the behaviour of synthetic genetic circuit itself and its impact on the host cell’s physiology [1,2]. It is necessary to have good standards to measure and represent accurately the biological parts, and to understand the interplay with the host’s cellular resources and functions. The conversion of a genetic sequence into a functional protein relies on the coordinated interplay of a multiplicity of biomolecules, which are provided by the host cell -at least until real orthogonallity is achieved [1].
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