Our Project: Context
Since the development of recombinant DNA technology (Cohen et al., 1973), recombinant protein production gradually became a major branch in modern biotechnologies bringing production of both biopharmaceutical and industrial enzymes to the unprecedented importance it reached today.
In the protein production process, the choice of a suitable host organism through several criteria is pivotal. In that regard, E. coli thanks to the availability of many molecular tools and the ease with which it is cultivated, remains an host of choice. Even though E. coli is a firmly established model organism, there is many remaining drawbacks to its utilisation. High among those, figures its inability to efficiently secrete protein into the growth medium but should also be noted that quite often high protein production induces E. coli’s stress response which hinders growth, and the very limited possibilities of post-translational modifications in E. coli.
In order to recover the biosynthesized product from an E. coli culture, bacteria must undergo lysis. This operation is expensive, inefficient, time consuming and implies that bioreactors must be restarted from scratch.
These problems drove a lot of researchers to the lab but it also drove the industrials away from E. coli and towards new host organisms.
The objective of our project is to reestablish E. coli as a microorganism of choice for recombinant protein production thanks to an easily implementable biobrick allowing regulable universal recombinant protein secretion.
Such biobrick could reveal itself to be a convenient alternative to cell lysis and be of great use in continuous mixed flow reactors for recombinant protein production and potentially many other applications. Moreover, cytotoxic protein production would also be greatly facilitated.
The idea behind The Gatekeeper is to create a porin regulated by inducers such as transition metal ions. To obtain this porin, we started from the pIV porin of the f1 bacteriophage. This porin allows the virion extrusion out of the bacteria without affecting its viability.
Through our semi-rational targeted mutagenesis approach, we should be able to quickly create high diversity pIV mutants libraries. Among those we aim to select the mutants for which we could affect the open/close state through the use of the previously mentioned inducers.
Check out this short explicative video about our project :
Cohen, S.N., Chang, A.C., Boyer, H.W.& Helling, R.B. (1973). Construction of biologically functional bacterial plasmids in vitro. Proc Natl Acad Sci U S A, Vol. 70, pp. 3240-3244