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From 2011.igem.org

Tools for Pathway Engineering

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The modular nature of synthetic biology makes it ideal for pathway engineering for the purpose of compound production. This means that engineered microorganisms can be used to manufacture a variety of useful chemicals, including pharmaceuticals and drug intermediates (e.g. Opiates), fine chemicals and biofuels.

When engineering a pathway, there are many considerations such as correctly inserting the pathway genes into your chosen host organism. However one of the most important considerations is the precise control of protein expression levels in order to control the carbon flux in the system.

If the protein is over expressing, it can have detrimental effects on the levels of cellular resources (such as levels of cofactors)and may even be cytotoxic. Overall yield is also affected by variance in levels of gene expression. This is why it is essential to ensure that gene expression over the whole pathway is optimal for the system.

One way of controlling the flux through pathways is to engineer the strength of promoters and ribosome binding sites (RBSs) to regulate gene expression. These can be used in a modular combinatorial approach to create diverse variations of the pathway of interest. To this end we have created a library of combined promoters and RBS of different strengths in biobrick format which can be used to create and test different expression levels of individual metabolic pathway genes for optimum product formation.

We have also created a Multiple Cloning site biobrick. It contains a double terminator and five restriction sites, so is ideal for use with our library of promoters. The aim of this biobrick is to significantly simplify the testing of genes by reducing the number of overall ligations that must be performed.

To read more about the creation of the Promoter + RBS library, click here.
To read more about the creation of the Multiple Cloning Site, click here.
To read more about potential modular pathways, click here.