Parts. Starting With The Basics: Standard Parts. The following parts are the basic components of our projects including the promoter, terminator, ribosome binding site and markers for our project. These parts are essentially the framework of our project. pSB1C3: A plasmid backbone with chloramphenicol resistance BBa_K2042004: Inducible Promoter BBa_B0014: Double Terminator BBa_J61100: Ribosome Binding Site Don’t Be Shy, Come On Out: Secretion System. A frame needs a canvas! These parts help create the type VIII secretion system necessary to bring our art to life! This is done through curli nanofiber biosynthesis using the parts below. CsgA-α (CsgA-Fibrin Knob Domain Fusion Protein): Stable Self-Assembling Monomer and CsgA-γ (CsgA-Fibrin Hole Domain Fusion Protein): Stable Self-Assembling Monomer: CsgA is the polymer forming unit of the curli biogenesis pathway which leads to biofilm formation in Escherichia coli. The wild-type csgA monomer can already self-assemble to form homopolymers outside of the cell. However, for our hydrogel art this interaction lacks sufficient stability. The mammalian protein fibrin arranges into fibrous chains to produce clots that hinder further blood loss. The polymerization of these chains relies on the interaction between N-terminal A- and B-knobs (alpha) and corresponding a- and b-holes (gamma) in the γ- and β-modules of fibrin [3]. When fused with protein csgA of the curli operon and chromoproteins, the fibrin knob domain (alpha) and the fibrin hole domain (gamma) will confer csgA the ability to stably crosslinking through the fibrin alpha and gamma domains. CsgC: Chaperone for secretion: csgC is a chaperone-like protein involved in the curli biogenesis pathway of gram-negative enterobacteria such as Escherichia coli. The curli biogenesis pathway results in the production of csgA nanofibers that allow bacteria to adhere to surfaces via biofilm formation. When the bacteria produces csgA, it is translocated to the periplasm in a Sec-dependent manner. There, csgC prevents the premature polymerization of csgA in the periplasm [4]. CsgE: forms secretion machinery with CsgG: csgE is an accessory protein in the Curli biogenesis pathway that allows for proper assembly of csgA subunits on the surface of gram-negative enterobacteria, such as Escherichia coli. Produced from the csgDEFG curli biogenesis regulatory operon, csgE localizes to the periplasmic space of E. coli. There, csgE acts as a chaperone protein by guiding the secretion of csgA through the outer membrane pore, CsgG, by forming a cap at the base of the pore[1]. By doing so, csgE can transport unfolded CsgA to CsgG for secretion [1]. CsgG: forms secretion machinery with CsgE: csgG is a key protein involved in the Curli biogenesis secretion-assembly pathway. As part of the csgDEFG operon, the csgG gene encodes for a peptide diffusion channel in the outer membrane of Escherichia coli [4]. Together with csgD, csgE, and csgF, csgG facilitates the proper secretion of curli subunits (i.e. csgA and csgB) from the periplasmic space. Once secreted, the csgA subunit (with the required assistance of csgB subunit) can aggregate and form amyloid fibrils at the cell surface Let There Be Color: Chromoprotein Parts. A canvas is boring without a little color! To bring our project to life, chromoproteins from iGEM’s 2011 Team Uppsala were utilized as the main form of bringing color to MicroMurals. Since the color is visible to the naked eye, they also serve as reporters for our plasmid expression. The following parts are the three chromoproteins we have chosen to use for our project. BBa_K864401: aeBlue, Blue Chromoprotein BBa_K1033914: amajLime, Lime-Green Chromoprotein BBa_K1033927: asPink, Pink chromoprotein The csgA-γ fusion sequence contains the γ fibrin hole domain downstream of the csgA sequence while the csgA-α fusion sequence contains the α fibrin knob domain upstream of the csgA sequence. Because of the location of fibrin relative to csgA in CsgA-γ and CsgA-α, we have chosen to fuse these three chromoproteins to CsgA-α rather than to both CsgA-α and CsgA-γ monomers.