The traditional motivation for integrating biological components into microfabricated devices has been to create biosensors that meld the molecular recognition capabilities of biology with the signal processing capabilities of electronic devices. However, a different motivation is emerging; biological components are being explored to radically change how fabrication is achieved at the micro- and nanoscales. Here we review biofabrication, the use of biological materials for fabrication, and focus on three specific biofabrication approaches: directed assembly, where localized external stimuli are employed to guide assembly; enzymatic assembly, where selective biocatalysts are enlisted to build macromolecular structure; and self-assembly, where information internal to the biological material guides its own assembly. Also reviewed are recent results with the aminopolysaccharide chitosan, a material that offers a combination of properties uniquely suited for biofabrication. In particular, chitosan can be directed to assemble in response to locally applied electrical signals, and the chitosan backbone provides sites that can be employed for the assembly of proteins, nucleic acids, and virus particles.

The cross-species bacterial communication signal autoinducer 2 (AI-2), produced by the purified enzymes Pfs (nucleosidase) and LuxS (terminal synthase) from S-adenosylhomocysteine, directly increased Escherichia coli biofilm mass 30-fold. Continuous-flow cells coupled with confocal microscopy corroborated these results by showing the addition of AI-2 significantly increased both biofilm mass and thickness and reduced the interstitial space between microcolonies. As expected, the addition of AI-2 to cells which lack the ability to transport AI-2 (lsr null mutant) failed to stimulate biofilm formation. Since the addition of AI-2 increased cell motility through enhanced transcription of five motility genes, we propose that AI-2 stimulates biofilm formation and alters its architecture by stimulating flagellar motion and motility. It was also found that the uncharacterized protein B3022 regulates this AI-2-mediated motility and biofilm phenotype through the two-component motility regulatory system QseBC. Deletion of b3022 abolished motility, which was restored by expressing b3022 in trans. Deletion of b3022 also decreased biofilm formation significantly, relative to the wild-type strain in three media (46 to 74%) in 96-well plates, as well as decreased biomass (8-fold) and substratum coverage (19-fold) in continuous-flow cells with minimal medium (growth rate not altered and biofilm restored by expressing b3022 in trans). Deleting b3022 changed the wild-type biofilm architecture from a thick (54-m) complex structure to one that contained only a few microcolonies. B3022 positively regulates expression of qseBC, flhD, fliA, and motA, since deleting b3022 decreased their transcription by 61-, 25-, 2.4-, and 18-fold, respectively. Transcriptome analysis also revealed that B3022 induces crl (26-fold) and flhCD (8-to 27-fold). Adding AI-2 (6.4 M) increased biofilm formation of wild-type K-12 MG1655 but not that of the isogenic b3022, qseBC, fliA, and motA mutants. Adding AI-2 also increased motA transcription for the wild-type strain but did not stimulate motA transcription for the b3022 and qseB mutants. Together, these results indicate AI-2 induces biofilm formation in E. coli through B3022, which then regulates QseBC and motility; hence, b3022 has been renamed the motility quorum-sensing regulator gene (the mqsR gene).There is an explosive amount of research on biofilms with the ultimate aim of their control (24); however, little is known about the regulation of this complex process of cell attachment leading to exquisite architecture (11). Since 65% of human bacterial infections involve biofilms (31), understanding the genetic basis of biofilm formation to find effective ways to prevent biofilms is important for combating disease and for engineering applications. To this end, we have studied the whole bacterial genome with DNA microarrays by two complementary approaches: studying biofilm gene expression relative to planktonic cells (34, 35) and studying plant-derived biofilm inhibitors that do not alter the bacterial g...

Bacterial autoinducer 2 (AI-2) is proposed to be an interspecies mediator of cell-cell communication that enables cells to operate at the multicellular level. Many environmental stimuli have been shown to affect the extracellular AI-2 levels, carbon sources being among the most important. In this report, we show that both AI-2 synthesis and uptake in Escherichia coli are subject to catabolite repression through the cyclic AMP (cAMP)-CRP complex, which directly stimulates transcription of the lsr (for "luxS regulated") operon and indirectly represses luxS expression. Specifically, cAMP-CRP is shown to bind to a CRP binding site located in the upstream region of the lsr promoter and works with the LsrR repressor to regulate AI-2 uptake. The functions of the lsr operon and its regulators, LsrR and LsrK, previously reported in Salmonella enterica serovar Typhimurium, are confirmed here for E. coli. The elucidation of cAMP-CRP involvement in E. coli autoinduction impacts many areas, including the growth of E. coli in fermentation processes.Bacteria have evolved complex genetic circuits to modulate their physiological states and behaviors in response to a variety of extracellular signals. In a process termed quorum sensing, or density-dependent gene regulation, bacteria produce, release, and respond to signaling molecules (autoinducers), which accumulate as a function of cell density. Quorum sensing allows bacteria to communicate with each other and coordinate their activities at a multicellular level. The autoinducers of many gram-positive bacteria are secreted peptides (30, 42), while gram-negative bacteria use small chemical molecules (60). Among gram-negative bacteria, the LuxI/LuxR signal synthase-signal receptor system is the most studied at the molecular level, with the signaling species being a family of N-acylhomoserine lactones. However, the cross-species autoinducer, autoinducer 2 (AI-2), has received intense interest recently because the gene for its terminal synthase, luxS, is present in over 55 bacteria and its activity can be readily assayed biologically (61). It is known that quorum sensing regulates diverse cellular processes, including bioluminescence (19, 34), spore formation (33), motility (18, 22), competence (35), conjugation (20), antibiotic synthesis (2, 17), virulence (38,44,50), and biofilm maturation (13, 45).Our laboratory is interested in understanding and controlling microbial behavior in bioreactors in order to enhance recombinant protein synthesis and yield. Since quorum sensing is emerging as a global regulator of many intracellular processes, including those that influence protein synthesis, efforts to understand this "tunable" controller are essential. In our previous work using chemostat cultures (14), many stimuli were found to affect the level of AI-2. Among these, the pulsed addition of glucose, a common carbon source for recombinant Escherichia coli fermentations, resulted in increased AI-2 levels, but with the dynamic response dependent on the steadystate growth rate (e.g., dil...