We thank other members of the Lyon INSA-ENS iGEM team (Viviane Chansavang, Mathilde Dumond, Alexandre Duprey, M&eacute;lanie Geffroy, Cl&eacute;mence Gonthier, Margaux Jaulin, Aur&eacute;lie Haag, Goki Ly, Thomas Poinsot, B&eacute;ryl Royer-Bertrand, Julie Soula, Michael Vonzy, Pierre Yves Zundel, Soufiane Bouhmadi, Olivier Brette, Ga&euml;l Chambonnier, Laura Izard, Aurianne Kroiss, Philippe Lejeune, Agn&egrave;s Rodrigue, Arnaud Rondelet, Sylvie Reverchon and Val&eacute;rie Desjardin), our sponsors for their financial support (bioM&eacute;rieux, Assystem, EDF, Fondation INSA, ENS-Lyon and the Department of Biosciences INSA-Lyon), F. Wisniewski-Dy&eacute; for critical reading of this manuscript and Dr C.C. Sze for strain gift. B. Drogue receives a Ph.D. fellowship from R&eacute;gion Rh&ocirc;ne-Alpes.

1. Biobrick Design and Synthesis of the Curli Operon
<ol>
	<li>Determine the genetic organization and localize the endogenous transcriptional and translational signals of the curli genes. These informations are gathered in specialized databases such as RegulonDBa or EcoGeneb and completed by a careful reading of pertinent publications. Data management and in silico preanalysis were performed with Clone Manager softwarec.</li>
	<li>Select the pertinent coding sequences. A set of...

<ol>
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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Escherichia+coli+curli+operons+in+directing+amyloid+fiber+formation.">Role of Escherichia coli curli operons in directing amyloid fiber formation.</a> Science. 295, 851-855 (2002).</li><li>Vidal, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+an+Escherichia+coli+K-12+mutant+strain+able+to+form+biofilms+on+inert+surfaces:+involvement+of+a+new+ompR+allele+that+increases+curli+expression.">Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression.</a> J. Bacteriol. 180, 2442-2449 (1998).</li><li>Hidalgo, G., Chen, X., Hay, A. G., Lion, L. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrillation+of+the+major+curli+subunit+CsgA+under+a+wide+range+of+conditions+implies+a+robust+design+of+aggregation.">Fibrillation of the major curli subunit CsgA under a wide range of conditions implies a robust design of aggregation.</a> Biochemistry. 50, 8281-8290 (2011).</li><li>Jubelin, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CpxR/OmpR+interplay+regulates+curli+gene+expression+in+response+to+osmolarity+in+Escherichia+coli.">CpxR/OmpR interplay regulates curli gene expression in response to osmolarity in Escherichia coli.</a> J. Bacteriol. 187, 2038-2049 (2005).</li><li>Ogasawara, H., Yamamoto, K., Ishihama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+biofilm+master+regulator+CsgD+in+cross-regulation+between+biofilm+formation+and+flagellar+synthesis.">Role of the biofilm master regulator CsgD in cross-regulation between biofilm formation and flagellar synthesis.</a> J. Bacteriol. 193, 2587-2597 (2011).</li><li>Mostaert, A. S., Higgins, M. J., Fukuma, T., Rindi, F., Jarvis, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+mechanical+characterisation+of+amyloid+fibrils+discovered+in+a+natural+adhesive.">Nanoscale mechanical characterisation of amyloid fibrils discovered in a natural adhesive.</a> J. Biol. 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No conflicts of interest declared.

Critical steps
The most critical step in this synthetic biology approach is the gene design. Synthetic gene design has to be meticulous to ensure an efficient system production. Two genes encoding the fiber monomers and three genes encoding proteins involved in their secretion system have been assembled with a strong and metal-inducible promoter to create a new functional unit for a novel application: the bio-decontamination of nuclear effluent. As planned and predicted, this device leads to...

Bacterial adherence to abiotic support plays a major role in bioremediation, biocatalysis or microbial fuel cells. Bioremediation processes use the capacities of microorganisms to degrade organic substances, or to modify the metal distribution (immobilization, volatilization) or speciation. These beneficial activities are observed in aquatic and terrestrial ecosystems, but also in the artificial systems developed to treat polluted water of industrial and domestic wastes. The intensity and the quality of the microbial activity depend on physico-chemical factors, but also on the lifestyle of microorganisms (free-floating or embedded into biofilm). The biofilm formation is associated with a metabolism promoting resistance to biocides by diverse mechanisms. This phenomenon will therefore be encouraged in most bioremediation processes. Moreover, engineering Escherichia coli cells to control the biofilm formation has been successfully applied to immobilize whole-cell sensors on biochips2-3.
Adaptation of microorganisms to high concentration of metals occurs via diverse mechanisms such as adsorption to extracellular matrix components, activation of efflux pump or specific carriers able to concentrate the metal into the cell. Boosting these bacterial activities via genetic engineering allows efficient and cheap treatment of metal pollution at the laboratory scale, especially in the case of highly toxic metals in weak quantity as described by Raghu et al. 2008 4. Bacterial remediation represents in this case a competitive and cost saving method compared to classical chemical processes using ion exchange resins. The authors described an E. coli chassis genetically engineered for cobalt uptake and retention first by knocking out the efflux pump encoding gene rcnA, and then by transformation with a multi copy plasmid allowing overproduction of a transporter with preferential uptake for cobalt. Such a strain appears as an efficient alternative to ion exchange resins to treat radioactive effluent, but a key unresolved issue is the recovery of contaminated bacteria at the end of the process 4. The objective of our work was therefore to engineer a custom-designed strain able to stick to abiotic supports such as glass or plastic.
Amongst the whole set of adhesins and adherent fimbriae identified in Gram- bacteria, we chose to design a system allowing curli production. Curli are thin (2-5 nm diameter) and highly aggregative amyloid fibers that protrude from the E. coli and Salmonella surface as a non-crystalline and insoluble matrix 5-7. Curli are also involved in the colonization of abiotic surfaces and the development of biofilms 8. Curli were recently shown to bind mercury ions 9. Amyloids are indeed known to possess high affinity for metals ions such as Cu2+, Zn2+ and Fe3+ 10. This property might further improve the decontamination of metal polluted effluents. The csg cluster is responsible for the production of curli fibers and is constituted of two divergently transcribed operons (Figure 1). The csgB, csgA and csgC genes constitute the sense operon, encoding the two curli subunits, CsgA and CsgB. CsgC seems to be involved in redox activity within the curli biogenesis system and to affect CsgG pore behavior 11. However, the absence of csgC in the majority of curli-producing bacteria indicates that the corresponding protein provides only a secundary level of control over the curli biogenesis. To simplify the system, we have been chosen to work with the minimum number of genes.
The csgDEFG operonencodes proteins essential in the regulation and transportation of CsgA and CsgB to the cell surface. CsgD is a transcriptional activator of the csgBAC operon and plays a key role in the control of biofilm formation by controlling the production of curli fimbriae and other biofilm components such as cellulose 12 and by inhibiting the flagellum production 13. CsgE, CsgF and CsgG constitute a curli-specific secretory apparatus in the outer-membrane through which the major curli subunit protein CsgA is secreted as a soluble protein. The polymerization of CsgA is dependent in vivo on the membrane-bound nucleator protein CsgB (reviewed in 14). Complex regulatory pathways involving several two-component systems have been shown to control curli gene expression 15-16. These complex regulations allow bacteria to form thick biofilms via the curli production in response to environmental cues, but are difficult to control for industrial applications. To facilitate the recovery of the metal-stuffed bacteria during an industrial process, bacterial fixation to a solid support indeed needs to be controlled by well defined parameter(s). The adherent properties of curli are linked to their amyloid nature 17 and could be used to improve bioremediation processes, but a simpler and easily controlled device has to be created.
Amongst these 7 genes 18, a set of 5 absolutely required genes for curli synthesis (csgB and csgA encoding fiber monomers) and export (csgE csgF and csgG, encoding the curli secretion complex) were selected to construct the synthetic operon. To escape the "natural" regulation of curli, a synthetic operon comprising these 5 csg genes under the control of a strong and cobalt-overinducible promoter (Figure 2) was designed and synthesized. The step-by-step analysis of the curli-encoding region and the design procedure for a functional synthetic operon are described. Two methods to visualize and quantify bacterial adherence to polystyrene and glass are explained.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>pIG2</td>
 <td></td>
 <td></td>
 <td>pUC57(pMB1 ori, 2710 bp) with a 3165 bp EcoRI/PstI fragment containing the synthetic Prcn-csgBAEFG operon ; Ampr </td>
 </tr>
 <tr>
 <td>pUC18</td>
 <td></td>
 <td></td>
 <td>Multicopy plasmid (pMB1 ori, 2686 bp), Ampr </td>
 </tr>
 <tr>
 <td>S23</td>
 <td></td>
 <td></td>
 <td>SSC1 (= GFP-tagged MG1655, gift of C.C. Sze)/pIG2</td>
 </tr>
 <tr>
 <td>S24</td>
 <td></td>
 <td></td>
 <td>SSC1/pUC18</td>
 </tr>
 <tr>
 <td>CoCl2</td>
 <td>Sigma</td>
 <td></td>
 <td>0,1M stock solution kept at Room Temperature</td>
 </tr>
 <tr>
 <td>M63</td>
 <td></td>
 <td></td>
 <td>29 </td>
 </tr>
 <tr>
 <td>24-well plate </td>
 <td>Nunc</td>
 <td>55429</td>
 <td>Polystyrene 24-well plates</td>
 </tr>
 </tbody>
</table>

engineering adherent bacteria by creating a single synthetic curli operon

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 1 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.

In silico annotation of the wild type csg sequence of E. coli K12 associated with the optimization of transcriptional and translational signals has allowed to design the single synthetic curli operon Prcn-csg shown in Figure 3 (full sequence in supplementary data). Protocol 2 and protocol 3 were used to visualize and quantify adherence associated with curli production. Using crystal violet staining on 24-well polystyrene plates (protocole 2) biofilm formation at...

Watch this Scientific Journal Video about Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon at JoVE.com

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

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14.3K vues.  Université de Lyon. La conception d'un encodage opéron synthétique à la fois l'appareil sécrétoire et les monomères de fibres structurelles curli est décrite. Surproduction de ces amyloïdes et les polymères adhérentes permet un gain mesurable de l'adhésion de l' E. coli Châssis 1. Une façon simple de visualiser et de quantifier l'adhérence sont expliqués.