title: Engineering adherent bacteria by creating a single synthetic curli operon.
abstract: The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011). The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control. The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.
authors.affiliation: UMR CNRS 5557 Ecologie Microbienne, Université Lyon 1, Université de Lyon.
authors.forename: Benoît
authors.initials: B
authors.lastname: Drogue
authors.forename: Philippe
authors.initials: P
authors.lastname: Thomas
authors.forename: Laurent
authors.initials: L
authors.lastname: Balvay
authors.forename: Claire
authors.initials: C
authors.lastname: Prigent-Combaret
authors.forename: Corinne
authors.initials: C
authors.lastname: Dorel
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics*; D001422:Bacterial Adhesion / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D048168:Escherichia coli K12 / Q000235:genetics*; D005813:Genes, Synthetic; D005818:Genetic Engineering / Q000379:methods; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D009876:Operon
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D049452:Green Fluorescent Proteins; C077481:Crl protein, Bacteria