| Summary: |
Bacterial biofilms are often regarded as a problem that can cause water contamination in potable distribution systems, infection or clogging of medical devices, contamination of household products and food preparations and biofouling of heat exchange systems. However, they can also be used advantageously, for example, in wastewater treatment and in the production of several commodities like organic acids and amino acids in biofilm reactors. A deeper understanding about the mechanisms underlying biofilm formation and stability will result in more efficient ways to either promote biofilm formation or to prevent their appearance. We aim to investigate the effect of recombinant protein expression and secretion in biofilm formation by Escherichia coli and Pseudomonas fluorescens. The cohesion of bacterial biofilms formed by these bacteria is dependent upon the secretion of extracellular polymeric substances (EPS) which are naturally secreted. E. coli strains have been engineered to secrete a recombinant fusion variant of Green Fluorescent Protein (GFP) from Aequoria victoria. The fusion protein contains two synthetic Z domains from Staphylococcal Protein A and the autofluorescent GFP moiety. This molecule, which has several clinical applications, will be secreted by the same pathways that are used for EPS secretion in E. coli and P. fluorescens. Since the transport capacity of these secretion systems is limited, a competition for secretion machinery components will exist between EPS and the recombinant protein. In the course of this project, several strains capable of secreting different levels of the recombinant protein will be assayed to analyse the competitive effect in secretion. These strains will harbour different plasmids of high or low copy-number and stronger or weaker promoters in order to obtain different expression and secretion levels. The recombinant protein may affect the dynamics of biofilm formation either by promoting aggregation or by decreasing EP  |
Summary
Bacterial biofilms are often regarded as a problem that can cause water contamination in potable distribution systems, infection or clogging of medical devices, contamination of household products and food preparations and biofouling of heat exchange systems. However, they can also be used advantageously, for example, in wastewater treatment and in the production of several commodities like organic acids and amino acids in biofilm reactors. A deeper understanding about the mechanisms underlying biofilm formation and stability will result in more efficient ways to either promote biofilm formation or to prevent their appearance. We aim to investigate the effect of recombinant protein expression and secretion in biofilm formation by Escherichia coli and Pseudomonas fluorescens. The cohesion of bacterial biofilms formed by these bacteria is dependent upon the secretion of extracellular polymeric substances (EPS) which are naturally secreted. E. coli strains have been engineered to secrete a recombinant fusion variant of Green Fluorescent Protein (GFP) from Aequoria victoria. The fusion protein contains two synthetic Z domains from Staphylococcal Protein A and the autofluorescent GFP moiety. This molecule, which has several clinical applications, will be secreted by the same pathways that are used for EPS secretion in E. coli and P. fluorescens. Since the transport capacity of these secretion systems is limited, a competition for secretion machinery components will exist between EPS and the recombinant protein. In the course of this project, several strains capable of secreting different levels of the recombinant protein will be assayed to analyse the competitive effect in secretion. These strains will harbour different plasmids of high or low copy-number and stronger or weaker promoters in order to obtain different expression and secretion levels. The recombinant protein may affect the dynamics of biofilm formation either by promoting aggregation or by decreasing EPS secretion due to a competition in protein secretion capacity. Thus the kinetics of biofilm formation may be different when comparing producing and non-producing strains. When mixed biofilms are promoted, population dynamics studies between producing and non-producing strains and between different species will be performed in order to find out which bacteria colonize the surface and how do the transitions between the immobilised and planktonic states take place. These experiments are particularly relevant due to their intrinsic competitive nature which is the dominant feature for most naturally occurring systems, as in environmental settings biofilms are rarely produced by single bacterial species. It is expected that biofilms containing recombinant proteins will have altered properties when compared to their natural counterparts thus the chemical and mechanical stability of these biofilms will be evaluated by shear stress treatment and the use of chemical biocides. |