Electricus aureus

BackgroundSalmonella typhimurium, a bacterium, within its genome contains a transcription activator called GolS. 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 attempts have been made to develop and characterise the gold sensing system of S. typhimurium. The activity of PgolTS and PgolB was analysed using fluorescent proteins RPF and GFP.

However, they faced some problems with basal expression levels of golS, which was causing some inconsistencies within their results.

The increasing interest in electricity production by microbes is reflected by the amount of research conducted and publications not only in scientific journals, but also in more public ones. For instance, Shewanella species bacteria are being extensively studied due to their unique cytochromes and ability to pump electrons through membrane, which are generated during respiration [5]. Specifically cytochromes mtrCAB from its genome were previously used in iGEM and have shown functioning in E. coli.

So we tried cloning the mtrCAB complex into pSB1C3 plasmid, unfortunately due to recurring nonsense mutations during assembly we did not have enough time to characterise the part. We did, however, submit a biobrick (BBa_K1127006) which contains all three mtrCAB open reading frames and rbs for mtrB & mtrC.

Bistability is commonly found in nature where resting is observed in either of two states (fig. 4). It usually involves a positive feedback loop accompanied by a sensitive regulatory step.These switches have capabilities to retain memories and make decisions, which has extreme biological importance [6].

We aimed to design a switch that is well regulated, tunable and robust. It must be bistable to synchronize current generation by the mtrCAB complex with gold mineralization by the peptides, as well as to manage resource allocation with respect to fitness of the whole bacterial population ( fig . 5 ) . We ended up submi t t i ng pa r t BBa_K1127016 but without having the time to characterise it.

Introduction

1. Gold sensing

Figure 1. Gold detoxification genes found in nature (A) and our designed construct (B).

Figure 2. β-galactosidase assay results. A - PgolTS+lacZα; B - golS.

2. Gold scavenging peptides

3. Electricity production

Figure 3. Schematic representation of mtrCAB functioning together with endogenous NapC protein.

4. The hybrid bistable switch

Figure 4. A simple representation of a bistable switch Figure 5. Illustration of a synthetic bistable switch for the expression on taR12. The genetic circuit is an extended version of quorum sensing from Vibrio fischeri. AiiA is included to disrupt the feedback loop caused by LuxI/R.

Microbial fuel cellThese devices are basically batteries engineered to exploit living cells to produce current, from the electrons produced during cellular respiration. Although they have already been documented in 20th century, novel genet ic engineering techniques have opened new ways of improving such systems.

However, traditional microbial fuel cells are bulky and inefficient. They are costly and less likely to be used on industrial scale. And so the need for miniaturized microbial fuel cells (mMFC) has grown extensively over the last decades. For example, Shogo Inoue and colleagues have demonstrated that MFC can be as small as the size of one cent US coin and still be able to function [7].

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 future (fig. 6). We would like to thank them and our other collaborators for their help and support during the project.

Figure 6. Miniaturized microbial fuel cell from team Bielefeld.

References

1. Checa et al. (2007) Bacterial sensing of and resistance to gold salts. Mol. Microbiol. 63, 1307-1318.2. Cerminati et al. (2011) Selective Detection of Gold Using GeneticallyEngineered Bacterial Reporters. Biotechnol. Bioeng. 108, 2553-2560.3. 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, 169-172.5. Jensen et al. (2010) Engineering of a synthetic electron conduit in living cells. PNAS USA. 107, 19213-19218.6. Jong et al. (2004) Qualitative Simulation of the Initiation of Sporulation in Bacillus subtilis. B. Math. Biol. 66, 261–299.7. Inoue et al. (2012) Structural optimization of contact electrodes in microbial fuel cells for current density enhancements. Sensors and Actuators A 177, 30–36.

Sponsors

In our design, we used lacZα gene to monitor the expression of our synthesized operon via β-galactosidase assay (fig. 1B), where we no longer have gold transportation system (golT) as it is not essential for the function of this operon.

ResultsTo determine activity of the gold sensing device, β-galactosidase assays were run in triplicates using PNPG as the substrate.

We demonstrated that E. coli DH5α transformed with BBa_K1127008 can respond to gold (fig. 2). The relative activity of β-galactosidase increased significantly in the presence of PgolTS and golS (DH5 alpha_AB) but was reduced to the basal level when golS was missing (DH5 alpha_A).

The results of Kruskal-Wallis were significant(X^2= 20.75, df = 7, p-value < 0.01).This signifies the function of golS as a gold-dependent transcriptional activator.

In all experiments, untransformed E. coli DH5α was used as our negative control and constructs were present within pSB1C3 plasmid.

BackgroundGold nanoparticles (AuNP) differ from solid particles by their chemical and physical properties and have characteristics making them useful in: medicine as drug delivery agents, chemical industry as a catalyst and various other areas. However, synthesis of AuNP with defined size involves toxic chemicals and therefore they cannot be used for biological applications.

Recently it was discovered that some bacteria like Delftia acidovorans is secreting small non-ribosomal peptides, which are able to reduce gold ions to solid gold, preventing the cell from damage. However, commercial synthesis of delftibactin is too expensive due to modified side chains of the compound.

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.

Our designWe decided to make different fusions with A3, Flg and MIDAS-2, tagging them with pelB signal sequences to facilitate their excretion. Also, due to small size of the peptides and potential degradation we also made fusions with IM9 tag (~9.5kDa).

Four parts were submitted to iGEM.We cloned the parts under constitutive promoter and used media supernatant for induction of AuNP formation and tried characterising them using Nanoparticle Tracking Analyzer (NanoSight LM10 ®). Unfortunately, the results did not provide evidence for gold bio-mineralization because changes were subtle and not significant compared to controls.

Over the past decades, mankind has been looking for a new source of energy. It must be green, clean and 100% renewable. Microbial fuel cells (MFC) can be considered as a plausible solution. However, MFC are inefficient and produces little electricity.

In order to improve the current design of MFC we aimed to introduce the following abilities through genetic modifications:1. The ability to sense gold in the environment2. The production of nanoparticles via short peptide secretion3. Regulated electron efflux through transmembrane protein complex4. Overal l system regulat ion in response to population viability and density.

Electricus 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.