Sarah Glaven3 Lina Bird1 Elizabeth Onderko1 Daniel Phillips2 Matthew Yates3 Christopher Voigt4

3, U.S. Naval Research Laboratory, Washington, District of Columbia, United States
1, National Research Council, Washington, District of Columbia, United States
2, American Society for Engineering Education, Washington, District of Columbia, United States
4, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States

It is well established that metal-respiring bacteria, such as Geobacter and Shewanella, perform extracellular electron transfer (EET) when grown as biofilms on the surface of electrodes. The electron transport (ET) proteins that enable this microbial electrical wiring in Shewanella have been identified and successfully expressed in E. coli, conferring an increase in the amount of current produced over the wild type background. The ability to rationally engineer EET processes in electrochemically active biofilms could result in leap-ahead technological advancements in biomaterials applications including microbial electrosynthesis, bioremediation, and microbial bioelectronics, specifically under austere conditions, such as the ocean. However, these applications are currently limited by a lack of understanding of the physiological constraints of the host bacterium (chassis) to properly and predictably express and orient ET proteins (e.g. c-type cytochromes) in the cell membrane, the ability to rapidly screen a large number of constructs for different ET pathways, and a library of operationally relevant chassis strains. In this talk I will describe results demonstrating the use of a suite of highly-optimized small molecule sensors (Marionette) developed for control over E. coli cellular processes to control expression of the Shewanella MtrCAB pathway, and accessory electron carriers, in Marinobacter atlanticus. First, Marionette sensors were transformed into M. atlanticus and assessed for expression of yellow fluorescent protein (YFP) after the addition of 7 different small molecules (choline, vanillin, naringenin, DAPG, cumate, tetracycline, and IPTG) during both planktonic growth and in the biofilm state. For most sensors, a broad dynamic range was observed similar to that demonstrated with E. coli when fluorescence was measured during log phase growth. Increasing fluorescence was also observed over time in biofilm associated cells as long as growth medium with small molecule inducer was continuously refreshed. When YFP was replaced with ET proteins, expression of MtrCAB led to an increase in current compared to the wild type strain when induced prior to inoculation into a bioelectrochemical system (BES). However, the effect was not robust enough for biosensing. Moving the MtrCAB pathway from a plasmid construct to the chromosome enabled more control over the quantity of protein expressed, however, no improvement in current was observed. When the same construct was tested in Shewanella oneidensis MR1 lacking the native MtrCAB pathway, current was found to be inducible following biofilm formation. Based on these results, we conclude that although the MtrCAB pathway can be successfully expressed in M. atlanticus, further optimization of export of these proteins to the outer membrane and/or connection to the inner membrane electron pool may be necessary. Understanding these constraints will advance the development of engineered bio-electrochemically active biofilms for development of self-healing living materials for energy and next generation electronics for the marine environment.