Team:TU Delft-Leiden/Human Practices/Electrace

ELECTRACE

In this section, we will first describe one of the areas of synthetic biology with the most promising outlook for applications: microbial biosensors. A number of criteria for a commercially successful microbial biosensor will be determined. We will then show that the ELECTRACE system we have developed meets these criteria. To do this, we will describe the application of our system in detail.

Microbial Biosensors

A biosensor is an analytical device which makes use of a biological component to detect an analyte [1]. This biological component is broadly defined and could include enzymes, nucleic acids, and antibodies. Here, we will focus on whole cell microbial biosensors. In whole cell biosensors, the biological component consists of a living cell [2]. The use of whole cell microbial biosensors has certain advantages over the use of other biosensors and non-biological sensors.

One of the major advantages of whole cell biosensors is that they are able to detect substances by class, and not only one specific substance [3]. Another advantage is that microbial biosensors can give not only analytical output (ie. what is the concentration of the analyte?) but also functional output (ie. how does my living system respond to the analyte?) [2]. In our discussion with Mr. Van Der Burg of BioDetection Systems , he mentioned in this context that with their whole cell biosensors, they stay close to the physiology of the subject to exploit this.

The biggest advantage of microbial biosensors however is the fact that evolution has already created an enormous amount of biosensors for us. Organisms sense environmental compounds to influence their decisions; they will only make a certain metabolic enzyme if the specific metabolite is sensed. Evolution has optimized such systems, thus creating bioreceptors for a lot of compounds [4]. This is used to find “new” biosensors: Mr Van Der Burg has told us that to find a dioxin bioreceptor, they “[search] for dioxin receptors which might have arisen in species living in contaminated areas.”

The most used sensing elements are promoter regions involved in the response of the cell to the target chemical. [3] With synthetic biology techniques, it is possible to isolate and enhance this promoter region and connect them to the appropriate reporter system. Currently, the most used reporters are genes which yield an optical (colorimetric, fluorescent, bioluminescent) signal. [3] These signals are processed with the use of various microscopy set-ups.

As already mentioned, nature has provided with a large array of possible microbial biosensors. With this as a start, a lot of whole cell biosensors have been developed. This is illustrated by the fact that only in iGEM, hundreds of biosensors have been developed. Also, D’Souza [4] published a list of 44 papers describing biosensors already in 2001. However, microbial biosensors have only very limitedly been commercialized. Su et al. [5] state: “Commercial microbial biosensors are just tips of the iceberg compared to the great amount of academic research on them”. They relate this to “the intrinsic disadvantages (slow response, low sensitivity, and poor selectivity)” of microbial biosensors, which, however, can and will be solved by the developments in synthetic biology [5].

One major reason microbial biosensors are currently limitedly used is not mentioned by Su et al., namely the fact that most current microbial biosensors require a microscopy set-up to be read out, due to the optical nature of their output. “For light and fluorescence assays you need lots of equipment and devious procedures,” Mr. Van Der Berg of BDS has explained to us. The equipment mentioned is very expensive: For instance, even the Zephyr DIY fluorescence microscope developed by iGEM team TU Delft 2013 costs around $1500, and this is already extremely cheap compared to commercially available machines. [6]

The need for bulky microscopy equipment and complicated procedures currently limits the use of whole cell biosensors to advanced laboratories with highly trained personnel. This greatly reduces the possibility for commercially viable applications. Whole cell biosensor-based consumer products are under the current circumstances impossible, but also business-to-business products would only be available for the small subset of companies which has (or can afford to pay for) a lab and technicians.

To increase the chances for commercial applications of microbial biosensors, outputs other than light-based outputs are needed. Several other output mechanisms are available. Bousse [2] mentions multiple methods to measure the energy metabolism of cells (including pH measurement and measurement of oxygen consumption). All these measurement require that the analyte has a distinct influence on the metabolism of the cell, for example due to toxicity or increased cell growth. This greatly limits the range of analytes which can be tested and also makes the construction of new sensors with synthetic biology techniques very difficult. Several other methods based on changing pH or redox potential have been described [7]. However, these methods are not widely used and much less popular than light-based outputs.

From these observations, it can be concluded that, in order to make a commercial application of a microbial biosensor possible, a novel output mechanism has to be developed. The novel output has to meet several requirements. Firstly, its output signal should be measurable with the use of equipment which is inexpensive, portable, and easy to handle by non-specialists. Secondly, the signal should be easily quantifiable: the relation between analyte concentration and output signal should be not too complicated. Thirdly, the output system should be modular. This means that different sensing elements (for example promoters) should be easy to connect to the output mechanism. Stated in a different way, the biosensor should have plug-and-play functionality.

Application

Our ELECTRACE system consists of bacteria which generate a current as a response to the measurement of an analyte. This current will be measured with a three-electrode setup and some circuitry called a potentiostat. This will be put together in a small handheld device.

The bacterial part will consist of E. coli cells which contain the BioBricks that make electron transport possible. The transcription and translation of the Mtr proteins, which form the transmembrane complex that makes extracellular electron transport possible, will be under control of a promoter sensitive to the compound we want to detect. In this way, the amount of ET complexes is directly coupled to the concentration of the analyte. Each of the ET complexes contributes to the overall extracellular electron transport. Therefore, the amount of transported electrons is directly related to the concentration of the analyte. To measure the amount of transported electrons, we have developed a gadget. This gadget consists of a microfluidic chamber in which the bacteria are placed, together with three electrodes. The extracellular electrons will reduce one of these electrodes, resulting in a current. This current will be measured with an integrated potentiostat. The outcome of a measurement (the current strength) is directly related to the amount of ET complexes and therefore the concentration of the analyte.

Inexpensive

The two main elements of the ELECTRACE system, namely the genetically modified bacteria, and the potentiostat circuitry, have great potential to be mass-produced. Mass production will make the system very inexpensive. Also, the novel technique of paper microfluidics provides an opportunity to make our device even cheaper. A very coarse approximation of the cost of an mass-produced ELECTRACE device would be around $10: $1 for the GMOs on a microfluidic paper strip, $4 for the potentiostat circuitry, and $5 for the casing. Although coarse, this price estimate clearly shows a price difference of about four orders of magnitude compared to a fluorescence microscope (without a lab, personel, etc.). Therefore, we could state that the ELECTRACE system is inexpensive compared to current methods.

Portable

The commercialized device as we envision it is very small. This will not be very hard to realize since all the major components (cells, circuitry, microfluidics) are small by themselves. Since the ELECTRACE system does not need a high voltage to work, a very small battery can be used. The fact that our device will be small will of course make it portable. This will provide great advantages for a range of applications. It should however be noted that at this stage of developmetn, we have not succeeded in scaling down the ELECTRACE system to a microfluids setup, as can be seen from the DropSens experiment data.

Easy to handle

To do a measurement with the ELECTRACE device, the only thing a user has to do is injecting an analyte in the microfluidic chamber. Doing this by hand is extremely difficult because applying a too high pressure damages the microfluidic system. To make this easier, some kind of small syringe pump has to be integrated in the system. This would make using the device fairly easy. Antoher way to make adding the analyte to the system easier is by making use of capillary forces: the device would then "soak up" the analyte.

Quantifiable

In current microbial biosensor systems, the concentration of analyte is coupled to the amount of emitted light. This light then has to be detected and converted to a current. This current is then measured to obtain a quantitative result. Our ELECTRACE system provides a direct coupling between the concentration of analyte and the current. In this way, we sidestep the optical step in the process, which will reduce noise. Also, the coupling between light-emitting proteins and detected light is highly non-linear and these kind of measurement are therefore dependent on complex calibration curves. [8] In the ELECTRACE system, the input/output relation will be much more simple. This makes quantification very easy compared to current microbial biosensor systems.

Plug-and-play functionality

The ELECTRACE system is very generic; the only thing specified for the sensing module is that it has to somehow activate a promoter. Therefore, essentially every biosensor promoter can be coupled to the electron transport module. In this way, our system is similar to the optical outputs currently used, of which the modularity is the biggest plus.

Since our system is developed using the BioBrick standards, our system can easily be combined with existing sensing promoter BioBricks. This provides the opportunity for our system to be widely used, tested and further characterized in the iGEM competition. This will speed up the optimization of our ELECTRACE system.

The modularity of the ELECTRACE system opens up the opportunity to develop a whole range of specific products based on the ELECTRACE system. In a sense, you could compare our ELECTRACE system to the operating system of your smart phone, and different biosensors based on ELECTRACE as smart phone applications. For mobile OS, open-source development is highly successful: open-source OS Android had 78.6 percent market share in 2013 [9]. Therefore, it is a logical step to further develop the ELECTRACE system in an open-source fashion, making the “source code” (the plasmids and the device designs) available for free. As a matter of fact, since ELECTRACE is developed as an iGEM project, we are already working open-source!

Issues

Unfortunately, even with all criteria discussed above fulfilled, commercial application of the ELECTRACE system is not as easy as it seems. We have hosted a discussion with a group of policymakers at the RIVM, during which we asked them: “How do we get from a working prototype to a commercial end product?” With their specialist input, we have been able to define the most important hurdles for commercial application: safety issues arising from outside-the-lab usage, and the possible discrepancy between open-source development and commercialization.

Usage out of the lab

Making the use of microbial biosensors (and synthetic biology in general) outside a lab environment possible has been one of the main goals during the development of the ELECTRACE system. In our opinion, development of outside-the-lab applications of synthetic biology will contribute to public understanding and acceptance of synthetic biology. It should therefore be the direction in which research should focus.

Under the current Dutch legislature [10][11], it is not allowed to use GMOs outside a carefully monitored laboratory environment. This makes the application of the ELECTRACE system as we have in mind currently impossible. In our discussion with policymakers discussion with policymakers, most of the attendants agreed that this legislature needs to be reconsidered.

The underlying reason for this strict legislature are safety concerns. Release of GMOs in the environment is generally considered very harmful. These concerns might be considered somewhat exaggerated, since almost every GMO is in essence a crippled version of lab strain which is not viable in nature. Next to that, introduction of parts such as kill switches might provide another safety measure to make a possible release in the environment less dangerous.

The safety risks for the ELECTRACE system are limited. Firstly, our GMOs will be immobilized in a microfluidics chamber; the bacteria will not be in contact with the environment. Next to that, adding the ET system reduces the viability of the cell, so our GMOs don’t have an evolutionary advantage. To further reduce the risk, a kill switch BioBrick could be implemented.

It is of course, even with aforementioned precautionary measures, possible that something goes wrong. However, one should ask oneself whether the benefits of using ELECTRACE, or synthetic biology in general, outside the lab outweigh the risks. In our opinion, this should be judged per application and usage outside the lab should not be prohibited beforehand.

Open-source development

Open-source development of the ELECTRACE system provides large opportunities to create a broad spectrum of applications and to optimize the system. However, there are also drawbacks for this development strategy. Firstly, open-source development requires very open communication about how the system is created and how it works. Therefore, ELECTRACE will inevitably be branded as a synthetic biology project. As a result of this, the applications will suffer from the negative framing concerning terms like SynBio and GMO. If the system is judged on the fact that it is synthetic biology and not on the usefulness of its applications, commercial success might be hampered.

Secondly, open-source development does not match with current revenue models of possible inventors and initial developers of the system. Since everything is freely available, companies can make their own ELECTRACE system to fit their application, and the inventors have nothing to sell. To create a fully functioning ELECTRACE system, a lot of research and development, and therefore money, is needed. There is a good chance commercial organizations will not be willing to spend this money when chances for future gains are negligible. A way to circumvent this problem would be to keep certain parts of the system “closed-source”. To get back to the analogy of smart phone OS, Google has kept certain parts of their Android closed or has closed them off again to make more money. [12] Another way to make more money would be not to patent the ELECTRACE system itself (then it wouldn’t be open source anymore!) but the use of the system in a certain way.

References

 

[1] Turner, Anthony; Wilson, George and Kaube, Isao (eds.) (1987). Biosensors:Fundamentals and Applications. Oxford, UK: Oxford University Press. p. 770

[2] L .Bousse, “Whole cell biosensors”, Sensors and Actuators B34 (1996) 270-275

[3] T. Vo-Dihn, B. Cullum, “Biosensors and biochips: advances in biological and medical diagnostics”, Fresenius J Anal Chem (2000) 366 :540–551

[4] S.F. D’Souza, “Microbial Biosensors” Biosensors & Bioelectronics 16 (2001) 337–353

[5] L. Su, Y. Lei, et al. “Microbial biosensors: A review” Biosensors and Bioelectronics 26 (2011) 1788–1799

[6] https://2013.igem.org/Team:TU-Delft/Zephyr (accessed on 30-9-2014)

[7] French CE, de Mora K, Joshi N, et al. “Synthetic biology and the art of biosensor design”. In: Institute of Medicine (US) Forum on Microbial Threats. The Science and Applications of Synthetic and Systems Biology: Workshop Summary. Washington (DC): National Academies Press (US); 2011. A5. Available from: http://www.ncbi.nlm.nih.gov/books/NBK84465/

[8] Young, IT, Vermolen, BJ, et al. “Photonic Calibration for Fluorescence Microscopy” (2008) Proc. of SPIE Vol. 6859 685915-1

[9] http://mobithinking.com/mobile-marketing-tools/latest-mobile-stats/a accessed on 1-10-2014

[10] Besluit genetisch gemodificeerde organismen milieubeheer (http://wetten.overheid.nl/BWBR0004703/geldigheidsdatum_30-09-2014#3) accessed on 30-9-2014

[11] Regeling genetisch gemodificeerde organismen (http://wetten.overheid.nl/BWBR0009653/Opschrift/geldigheidsdatum_30-09-2014) accessed on 30-9-2014

[12] http://arstechnica.com/gadgets/2013/10/googles-iron-grip-on-android-controlling-open-source-by-any-means-necessary/ accessed 1-10-2014