Synthetic Biology Tools for the Membrane – Targeted Localisation and Elucidation of Protein Interactions

To meet the need for new, green production scenarios, development of biological cell factories is becoming increasingly important. In order for cell factories to compete with traditional production means, it is essential to expand the available toolbox. We are developing tools for the E. coli cell factory, focusing on the membranes. Surface displaying enzymes may allow for development of whole-­‐cell catalysts, but a recurring issue with the technique is to know that a protein is indeed surface displayed, and not detected due to lysis of cells or secretion. We are developing a fluorescence-­‐based method for easy detection of surface display using GFP-­‐nanobodies (1). We fused a GFP-­‐nanobody to the E. coli outer membrane anchor LppOmpA, resulting in surface display of the nanobody. Addition of free GFP to the whole cells resulted in binding of GFP to the cells via the nanobody, as shown by fluorescence measurements, in-­‐gel fluorescence and microscopy (fig. 1). We are now further expanding the toolbox for use in surface display applications. In addition, to expand our knowledge of protein interactions and protein complex formation in the membrane, we are exploring the use of styrene maleic acid (SMA) for isolation of membrane proteins. SMA is a polymer which spontaneously digs into a lipid membrane and carves out a disc containing protein and native lipids (2). By elucidating protein interactions we will be able to tune and optimise heterologous pathway expression in our E. coli cell factories.