Design and characterisation of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology
Synthetic Biology is the design and construction of novel biological parts, devices and systems, as well as the redesign of existing biological systems for useful purposes (http://syntheticbiology.org). The basic principles of Synthetic Biology are: (i) the use of standardised and well characterised building blocks, (ii) the hierarchical design of nature-inspired, artificial genetic circuits and proteins in silico, and (iii) the use of chemically synthesized DNA sequences not found in nature. For the rational design of artificial genetic circuits the behaviour of each individual part of the circuit has to be well characterised. Promoters are key parts for the heterologous expression of proteins. The vision of cyanobacterial synthetic biology is to build up a repository of standard genetic parts shared among the scientific community to speed up the successful genetic/metabolic engineering for the sustainable production of valuable products.
Regardless of all the efforts to characterize the promoter and understand its behavior in different conditions, it is clear that it will still be dependent on the cell transcription machinery. Therefore, undesired cross-talk between various transcriptional networks may be too difficult to insulate, resulting in unexpected expression patterns and leading to abnormal metabolic flux. Consequently, orthogonal promoters are required to control the expression of genes when metabolic pathways are to be introduced or modified. As an example, the hybrid promoters tac and trc have been used to regulate the expression of various genes in cyanobacteria. However, these well described promoters used in Escherichia coli have been shown to respond differently in a cyanobacterial background, supporting the notion that all these parts have to be initially and thoroughly characterized in cyanobacteria.
The principal behind Synthetic Biology is to, in a standardized engineering manner, design and construct a cell with a desired function and metabolism. It is a molecular approach using standardized artificially synthesised genetic building blocks (so called BioBricks; e.g. promotors, RBSs, genes, transcription stops, degradation tags) in a multipurpose cloning vector. The chemical DNA-synthesis allows the production and use of DNA sequences not found in nature. The individual specific building blocks are easily exchanged for others using the standardized BioBrick interface.
To enable the development of Cyanobacterial Synthetic Biology, we have previously developed and characterised e.g. several molecular tools: (i) a broad-host-range BioBrick shuttle vector constructed and confirmed to replicate in E. coli and in cyanobacteria, (ii) reporter genes, (iii) RBSs, (iv) promoters (constitutative and regulated), and (v) protein degradation tags.
At present we are further developing wide-dynamic range regulated promoters to be used when engineering cyanobacteria for specific purposes.
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