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iGEM York

iGEM York Team

Overview

Over the past decades, mankind has sought for a new source of energy. It must be green, clean and 100% renewable. Microbial fuel cells (MFC) come to us as a plausible solution. The concept of tiny organisms converting waste materials into electricity has captured attention of scientists and publics around the world. However, MFC is inefficient and produces little electricity. In this project, we aim to improve MFC through decomposition of gold nanoparticles within the anodic chamber. What is more, our MFC must be bistable as well as tunable to work well under a wider range of conditions.

We engineer the genetic circuit of Escherichia coli, such that the bacteria could sense gold and respond accordingly. Our circuit is subdivided into three modules (Figure 1). The first is gold sensing. It is designed to recognize gold (HAuCl4) in its environment. GolS is a transcriptional activator of the promoter P(golTS). Upon binding with the gold, it dimerizes and binds to the recognition site within the promoter. The second is gold scavenging peptides. There are four variants of the fusion between MIDAS-2 and A3. We also include the N-terminal pelB tag for secretion to the periplasm and IM9 for higher stability. Note that the tag is likely to be removed during transportation process and potentially our peptides should be released to the extracellular space. IM9 tag is relatively small (~9.5 kDa) and should not interfere with mineralization mediated by the peptides. Genes for the peptides are placed downstream of the gold sensing promoter, such that mineralization is conditional to concentrations of gold in the environment. The final module is hybrid bistable switch.

The switch is an extended version of the quorum sensing from Vibrio fischeri. LuxI forms an indirect positive feedback loop through interactions with LuxR which is expressed by the constitutive promoter. Upon gold induction, AiiA is expressed and degrade the quorum sensing signal AHL. In the other word, AiiA could disrupt the feedback loop. When gold is absent and population density is high, taR12 which is downstream of the Plux/lac is upregulated. In our project, taR12 is to transactivate the translation of mtrA upon binding to the crR12. Electricity is generated when all three mtr proteins are expressed and translocated to the right locations in cells.

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Gold sensing

Some bacteria evolved to sense various toxic metals in the environment and produce certain responses. It involves transcription factors, different promoters and cascade activation reactions. One of the most characterised transcription factors belongs to MerR class. They are responsible for responding to stresses like antibiotics or heavy metals and the mechanism involves conserved N and C-terminal domains which contain DNA and metal binding motives (1, 2). E. coli has a MerR like transcriptional activator CueR, which allows the bacteria to sense copper ions and activate the transcription of specific genes. The same transcription factor can also allow the bacteria to sense the presence of gold ions in the environment (3).

There are few problems involving CueR transcription activator for gold sensing. First of all; it is not entirely selective for gold ions and copper as well as silver ions can be involved in the activation of response genes. Secondly; it is a naturally present regulator of 2 genes in E. coli (4), therefore its use would involve some unsuspected behavior- we could not measure the exact response. The solution might involve mutating these 2 genes (which requires a lot of time) or finding alternative regulator system.

S. typhimurium also contains a MerR like transcription activator GolS (6). 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) (7). The deletion of these genes was shown to affect the bacteria in the presence of gold and increase mortality. Additionally, GolS is also able to control some other efflux systems, however it requires much higher levels than for the gol regulon and therefore would not be very sensitive for quantitative studies (9). By using this knowledge several attempts have been made to develop and characterise the gold sensing system from S. typhimurium (5, 8). Some of them involved PgolB and some PgolT/S promoters where the most common response molecule fluorescent proteins like RFP or GFP (Fig 1b). However, they faced some problems with basal expression levels of golS, which was causing some inconsistencies with their results.

Fig 1. Gol regulon found in S. typhimurium with all of the required components for detoxification system (a). The synthetic gol regulon with reporter molecule RFP under PgolB promoter (b).

We decided to use lacZ as a response molecule and make a gold sensing device containing PgolT/S-lacZ-golS in a standard iGEM plasmid. Also, the lacZ gene would have two restriction sites around it so it can be removed and exchanged with another response molecule or a response molecule that would transfer the signal to further downstream elements. The device would look like synthetic gol regulon Fig 1B, however the PgolB and RFP will not be present and between PgolT/S and golS the “replaceable” (SacII and SanDI restriction enzymes) lacZ will be present.

Two linear fragments were ordered as gBlocks containing PgolT/S-lacZ and golS with XbaI and SpeI restriction sites on the sides. However, later on we had to change the restriction sites by PCR, because the plasmids were only re-ligating during the cloning process and we were unable to transfer the genes and fuse these two parts together. Finally, a PgolT/S-lacZ-golS construct was made into pSB1C3. The activity was tested on LB plates containing X-gal as well as a -galactosidase assay to best understand the response time and required gold ion concentration for induction.

Biobrick	Short name	Length	Characterized
BBa_K1127008	PgolTS+LacZ+GolS	754 bp	Yes

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Results

Gold scavenging peptides (BBa_K1127000-3)

This device is constructed to recognize gold (HAuCl4) in its environment. GolS is a transcriptional activator of the promoter P(golTS). Upon binding with the gold, it dimerizes and binds to the recognition site within the promoter. We also include LacZ alpha that is useful for quantification of the device activity via Beta-galactosidase assay. The mechanism is simplified and shown in Figure 1.

To determine activity of the gold sensing device, beta-galactosidase assays were run in triplicates using PNPG as the substrate. In all experiments, untransformed E. coli strain DH5 alpha or strain BL21 were used as our negative control. The protocols are described in more detail in the Protocols section.

We demonstrated that E. coli strain DH5 alpha transformed with BBa_K1127008 can respond to the gold. In Figure 2a, relative activity of the enzyme beta-galactosidase increased significantly in the presence of golS and promoter P(golTS) (DH5 alpha_AB) but reduced to the basal level when golS was missing (DH5 alpha_A). The results are well supported by statistics - Kruskal-Wallis X^2= 20.75, df = 7, p-value < 0.01. This signifies the function of golS as a gold-dependent transcriptional activator.

In Figure 2b, E. coli strain BL21 has background expression of the enzyme. Strong enzyme activities were observed in the absence of lacZ alpha (BL21 and BL21_B), so that no differences were observed (Kruskal-Wallis X^2= 6.69, df = 3, p-value > 0.05). This suggests that there could be beta-galactosidase in the chromosome of this E. coli strain. Further research has confirmed our hypothesis. Beta-galactosidase (accession no. = C6ELN6) is present in BL21 according to the gene database on uniprot.org.

We determined kinetics of the device. DH5 alpha were exposed to different gold concentrations for 16 hours to reach steady states. We found that the response was non-linear with distinct phases - exponential phase (0.0uM - 2.5uM AuCl4), stationary phase (2.5uM - 10.0uM AuCl4) and logarithmic decline phase (not shown). The data were plotted in Figure3a with simulation of the fitted logistic model. Please visit our Modelling section for more detail. As seen in the plot, the device is very sensitive to the gold as low as 0.5uM. Later on, it became saturated from about 10uM onwards. The saturation could be caused by several factor, such as maximum promoter activity, expression and stability of the proteins and diffusion limit of the gold. In Figure 3b, we ran more assays on the subpart P(golTS)+LacZ alpha to confirm the function of golS. It is obvious that the promoter alone can't respond to the gold (Kruskal-Wallis X^2= 4.68, df = 6, p-value > 0.5).

E. coli strain DH5 alpha were transformed with the device or one of the subparts. They were streaked on the LB agar plates supplemented with chloramphenicol and Xgal. If LacZ alpha is expressed, the enzyme will degrade the substrate Xgal and turn colonies blue. In Figure 4, the colonies appear blue apart from those on the DH5 alpha+golS plate. This supports our hypothesis that P(golTS) is the minimal unit for the expression of LacZ alpha.

Gold scavenging peptides (BBa_K1127000-3)

We created four variants of the fusion between MIDAS-2 and A3. We also include the N-terminal pelB tag for secretion to the periplasm and IM9 for higher stability. Note that the tag should be removed during transportation process and potentially our peptides should be released to the extracellular space. IM9 tag is relatively small (~9.5 kDa) and should not interfere with mineralization mediated by the peptides.

For peptide activity testing , we cloned the peptides under the constitutive promoter (BBa_J23100). Characterization using Nanoparticle Tracking Analyzer (NanoSight LM10 ®). The device allows us to visualize, measure and characterize any type of nanoparticles and any size down to 10nm. The technology is based on optical properties as well as Brownian motion of the particles. Please visit http://www.nanosight.com/ for more information.

We obtained a series of recordings and several analytical reports of the particles within samples from the overnight cultures of E. coli strain DH5 alpha transformed with the hybrids. Unfortunately, the results can't provide evidence for gold bio-mineralization because changes were subtle and not significant compared to the controls (untransformed E. coli). Please visit out Protocols section for more detail.

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Safety

Safe Practices in the Lab

Our team conducted the research at the University of York, in the teaching laboratories of the Biology Department. All work was conducted in a biosafety level (BSL) 1 laboratory. We used Escherichia coli DH5a, BL21 and K12 wild type, which are well characterised and do not constitute a threat to the environment. Shewanella oneidensis and Salmonella enterica typhimurium serovar 14208s were not used (as we had the genes synthesised, instead of amplifying them from the genome). Before gaining access to the lab space, team members were required to:

Complete chemical waste disposal training
Attend an orientation with Dr Jen Lee and Nikki Begg, part of the technical staff of the labs, regarding the University of York Health & Safety Regulations
Receive specific training from Dr Jen Lee and from Dr James Chong, our main supervisor, for the equipment in the lab
Complete a risk assessment form provided by Biological Safety Advisor (David Nelson)
Sign after-hours access agreements with the University, after having been instructed on the regulations and procedures which apply for working in the lab at weekends and between 18:00-08:00

Our standard lab practices were in compliance with the World Health Organization’s Biosafety Level 1 guidelines. Safety forms were approved on September 22, 2013, by Evan Appleton of behalf of iGEM. Many of our basic lab practices are described below.

Researchers were required to wear gloves while in the lab space, and to remove both gloves and wash their hands when going into the write-up area or in other lab common areas;
No gloves were allowed to leave the lab space, and no food or drink was allowed into the lab space, including the computer room;
Within the Department of Biology, microbiological samples were transported only via the internal service elevator and on lab-associated stairs which were accessed by card, to avoid contamination of public areas;
All flammable liquids were kept in a flammable storage cabinet;
The lab contained distinct waste containers for general waste, biohazard waste, biohazard sharps, and non-biohazard broken glassware;
Liquid bacterial waste was always autoclaved before being discharged into sanitary sewage;
Benchtops were decontaminated with ethanol before and after lab work was conducted;
Tools that came into contact with bacteria were soaked in ethanol and flame-treated before and after use;
An autoclave was used to decontaminate growth media, glassware, tubes, pipette tips, etc. All lab members were trained in preparing waste and media for being autoclaved. The actual autoclaving programs were run for us by the staff of the teaching labs, to avoid dangers to researcher safety;
An emergency shower, eyewash, and first aid kit were available within the lab space in case of emergency.
Dry ice was carried in iceboxes and thick gloves were used to take samples from them. Dry ice was left in the room to evaporate instead of disposing anywhere.
Microwave was used for melting agarose for agarose gels and LB agar for making LB agar plates. Thick gloves were used to take hot samples after heating.
Gel electrophoresis tanks were kept separately to avoid any electric shock.
All the unused isolated DNA was autoclaved in order to shear the genetic material that would not be used by bacteria in environment.
While using Bunsen burners no gloves were worn in order to prevent melting the gloves and causing high level of damage to the skin.
For adjusting the pH highly corrosive 1M NaOH was used. Thus gloves and protective glasses were worn in order to avoid any skin burning.
Phenol/Chloroform extraction of S. oneidensis genome was performed in the fume hood as both of the compounds are volatile and toxic.
β-mercaptoethanol was used during the beta galactosidase activity assay. All the procedures performed in the fume hood to avoid intoxication of highly volatile compound.
As gold ions are toxic to most of the living organisms after each experiment we treated them with the excess of HEPES to form gold aggregates as well as washed with excess of water to dilute to non-toxic concentrations.

For more information please refer to the safety forms:
Laboratory Safety Guidelines
Laboratory Safety Form
Safety forms were approved on September 22, 2013 by Evan Appleton.

To minimise the risk one might be exposed to when working with Ethidium Bromide (which is a carcinogen) we used its safer alternative, SYBR Safe, for our gel electrophoresis. Team members were protected from exposure to UV rays during gel reading with a UV shield. Facial shields were also available for use when needed.

Safety Concerns for Our Project:

There are no serious safety concerns, as the genes or their products that we work on have no identified risk to human health, do not influence the environment and are specific for only our project.

Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?

The only safety issue associated with our new BioBrick parts is researcher safety in testing the parts. We have documented our standard operating procedures above, which other teams can reference if they want to use our parts, or test other parts using arsenic or naphthalene. We are also conducting extensive tests to assess the functionality of our BioBrick parts, to ensure that they behave as expected under different conditions.

Safety Concerns for iGEM Competition:

Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

use other selection forms like auxotrophic mutants for amino acids, not antibiotic resistance, to prevent release of resistant strains into the environment. However, because we wanted to try MFC with wastewater, this method would not be useful as in addition we would need to add amino acid, which would affect the metabolism.
usually the antibiotics that are used in the lab are different from medically common antibiotics and would potentially cause no influence towards MRSA and similar
make standard iGEM safety guidelines to be followed by all the teams