ElectricusElectricus aureus aureus: our greatest source of power comes from the smallest organisms on Earth

Bucaite, G., Canavate, R., Duangrattanalert, K., Farthing, A., Gyulev, I., Johnson, H., Klumbys, E., Kondratavicius, J., Miles, R., Necula, A., Panayotov, N., Pearson, C., Petrovs, R., Rizzo, A. Supervisors: Dr Chong, J., Dr Edwards, J., Prof Smith, M., Dr Thomas, G.

Introduction 3. Electricity production

Over the past decades, mankind has The increasing interest in electricity production by been looking for a new source of microbes is reflected by the amount of research energy. It must be green, clean and conducted and publications not only in scientific 100% renewable. Microbial fuel cells journals, but also in more public ones. For instance, (MFC) can be considered as a plausible Shewanella species bacteria are being extensively solution. However, MFC are inefficient studied due to their unique cytochromes and ability to and produces little electricity. pump electrons through membrane, which are generated during respiration [5]. Specifically In order to improve the current design of cytochromes mtrCAB from its genome were previously MFC we aimed to introduce the used in iGEM and have shown functioning in E. coli. following abilities through genetic Figure 3. Schematic representation of mtrCAB So we tried cloning the mtrCAB complex into pSB1C3 modifications: functioning together with endogenous NapC protein. plasmid, unfortunately due to recurring nonsense 1. The ability to sense gold in the mutations during assembly we did not have enough time to characterise the part. We did, however, environment submit a biobrick (BBa_K1127006) which contains all three mtrCAB open reading frames and rbs for 2. The production of nanoparticles via mtrB & mtrC. short peptide secretion 3. Regulated electron efflux through transmembrane protein complex 4. Overall system regulation in 4. The hybrid bistable switch response to population viability and density. Bistability is commonly found in nature where We aimed to design a switch that is well regulated, resting is observed in either of two states (fig. 4). It tunable and robust. It must be bistable to usually involves a positive feedback loop synchronize current generation by the mtrCAB 1. Gold sensing accompanied by a sensitive regulatory step.These complex with gold mineralization by the peptides, switches have capabilities to retain memories and as well as to manage resource allocation with make decisions, which has extreme biological respect to fitness of the whole bacterial population Background importance [6]. ( fi g . 5 ) . We e n d e d u p s u b m i t t i n g p a r t Salmonella typhimurium, a bacterium, within its BBa_K1127016 but without having the time to genome contains a transcription activator called GolS. characterise it. It is responsible for expression of the gold detoxification system involving the transcription of golB (gold ion binding protein) from the promoter PgolB as well as golS (transcription of itself gene) and golT (gold ion transportation system) transcription from promoter PgolT/S (fig. 1A) [1].

In previous studies [2] the deletion of this regulon was shown to have an effect on bacterial mortality. Several Figure 1. Gold detoxification genes found in nature (A) attempts have been made to develop and characterise and our designed construct (B). the gold sensing system of S. typhimurium. The activity of PgolTS and PgolB was analysed using fluorescent In our design, we used lacZα gene to monitor the proteins RPF and GFP. expression of our synthesized operon via β- galactosidase assay (fig. 1B), where we no However, they faced some problems with basal longer have gold transportation system (golT) as expression levels of golS, which was causing some it is not essential for the function of this operon. inconsistencies within their results.

Results Figure 4. A simple representation of a bistable switch Figure 5. Illustration of a synthetic bistable switch for the expression on taR12. The To determine activity of the gold sensing device, genetic circuit is an extended version of β-galactosidase assays were run in triplicates using quorum sensing from Vibrio fischeri. AiiA is PNPG as the substrate. included to disrupt the feedback loop caused by LuxI/R. We demonstrated that E. coli DH5α transformed with BBa_K1127008 can respond to gold (fig. 2). The Microbial fuel cell relative activity of β-galactosidase increased significantly in the presence of PgolTS and golS (DH5 These devices are basically batteries engineered alpha_AB) but was reduced to the basal level when to exploit living cells to produce current, from the golS was missing (DH5 alpha_A). electrons produced during cellular respiration. Although they have already been documented in The results of Kruskal-Wallis were significant 20th century, novel genetic engineering (X^2= 20.75, df = 7, p-value < 0.01). techniques have opened new ways of improving This signifies the function of golS as a gold-dependent such systems. transcriptional activator. However, traditional microbial fuel cells are bulky In all experiments, untransformed E. coli DH5α was and inefficient. They are costly and less likely to be used as our negative control and constructs were used on industrial scale. And so the need for miniaturized microbial fuel cells (mMFC) has present within pSB1C3 plasmid. grown extensively over the last decades. For Figure 2. β-galactosidase assay results. example, Shogo Inoue and colleagues have A - PgolTS+lacZα; B - golS. demonstrated that MFC can be as small as the size of one cent US coin and still be able to function [7].

2. Gold scavenging peptides The iGEM Bielefeld team has provided us with their designed MFC with a minimalistic total volume of 15.8 cm3, which we hope to use in the Background Our design future (fig. 6). We would like to thank them and our Gold nanoparticles (AuNP) differ from solid We decided to make different fusions with A3, Flg other collaborators for their help and support during the project. particles by their chemical and physical properties and MIDAS-2, tagging them with pelB signal Figure 6. Miniaturized microbial fuel cell from team Bielefeld. and have characteristics making them useful in: sequences to facilitate their excretion. Also, due to medicine as drug delivery agents, chemical small size of the peptides and potential industry as a catalyst and various other areas. degradation we also made fusions with IM9 tag However, synthesis of AuNP with defined size (~9.5kDa). References involves toxic chemicals and therefore they cannot Four parts were submitted to iGEM.We cloned the be used for biological applications. 1. Checa et al. (2007) Bacterial sensing of and resistance to gold salts. Mol. Microbiol. 63, 1307-1318. parts under constitutive promoter and used media 2. Cerminati et al. (2011) Selective Detection of Gold Using GeneticallyEngineered Bacterial Reporters. Biotechnol. Bioeng. 108, Recently it was discovered that some bacteria like supernatant for induction of AuNP formation and 2553-2560. Delftia acidovorans is secreting small non- tried characterising them using Nanoparticle 3. Kim et al. (2010) Peptide mediated shape and size tunable synthesis of gold nanoparticles. Acta. Biomater. 6, 2681-2689. 4. Naik et al. (2002) Biomimetic synthesis and patterning of silver nanoparticles. Nat. Mater. 1, 169-172. ribosomal peptides, which are able to reduce gold Tracking Analyzer (NanoSight LM10 ®). 5. Jensen et al. (2010) Engineering of a synthetic electron conduit in living cells. PNAS USA. 107, 19213-19218. ions to solid gold, preventing the cell from damage. Unfortunately, the results did not provide evidence 6. Jong et al. (2004) Qualitative Simulation of the Initiation of Sporulation in Bacillus subtilis. B. Math. Biol. 66, 261–299. for gold bio-mineralization because changes were 7. Inoue et al. (2012) Structural optimization of contact electrodes in microbial fuel cells for current density enhancements. However, commercial synthesis of delftibactin is Sensors and Actuators A 177, 30–36. too expensive due to modified side chains of the subtle and not significant compared to controls. compound. Sponsors

Alternatives such as A3 or MIDAS-2 gold binding peptides were discovered [3,4]. These are short dodecamer peptides which have shown to coat AuNP and prevent them from aggregation. A3 derived peptides need and external gold reducing agent (HEPES buffer), whereas MIDAS-2 is able to reduce gold on its own.