• Choose a method of cell immobilisation
• Design final biosensor based on consumer needs
• Initial sketches were done.
• iGEM Hamburg rendered our plate and 3D printed it for us.
• A latex nanoporous coating method on a paper base was chosen.
• A fruit sticker, industrial sticker, and plate design were all chosen as the final designs.
• Biosensor strips will be biodegradable. • Interactions with other systems evaluated.
KEEP FRESH embodies the final biosensor device. The SENSE and EXPRESS elements were first assembled in a plasmid so the function of the overall biocircuit could be tested. Following this, various engineering considerations such as selectivity and specificity were taken into account, along with government policies surrounding GMOs, to design a feasible product with viable real-life applications. APPLIED DESIGN follows the technology and design selection process, and evaluates the impact of the biosensor on the environment.
Circuitry Once the SENSE and EXPRESS elements of our project were optimised, the final task was to assemble them together as a final construct to create the biosensor circuitry. The genetic function of the biosensor acts like a circuit, as can be seen in the plasmid diagram below.
Activation of the biosensor occurs when the SENSE elements are switched on in the presence of ethylene. EtnR2 binds to ethylene and causes the phosphorylation of EtnR1. This complex binds to promoter DNA to also activate transcription of downstream DNA. In this circuit, the EXPRESS part of the system encapsulates this downstream segment. The expression of amilCP causes a visually perceivable colour change from white to blue. KEEP FRESH uses this circuit as the core of the biosensor device.
Scalability The initial prototype or production of this biosensor must be easily scaled up for widespread use over time. Key factors to consider include the upscaling of cells required to produce the mechanism required. Bioreactor sizing is crucial, and having successive reactor tanks of increasing size will allow for a large batch of cells to be grown under optimal conditions in order to meet a larger scale production requirement.
The technology should also consider scale of application. This technology has potential uses for an individual consumer at a household level as well as industrial or quality assurance applications for large produce players all the way up the supply chain. By designing a biosensor that can take many forms, a wide scale of users can be catered for.
Selectivity and specificity Selectivity refers to the competition of various molecules to a receptor, whilst specificity refers to the unique activation of a receptor by a certain molecule, and they go hand-in-hand in biosensor design. Many sensing devices do not respond to one sole molecule, but rather to a feature of that molecule. For example, a common infrared carbon monoxide sensor operates on the function that doubled-bonded carbons absorb energy of a particular wavelength. It would, however, pick up a signal from other compounds that also contain a double bond attached to a carbon (Sberveglieri 2012). Other sensors may instead be specific to a metabolite of a molecule of interest rather than the molecule itself, or having competing selectivity to multiple compounds of a similar physical structure. The sensitivity and specificity of a biosensor is one of the pillars of its characterisation.
Recyclability Waste is another particularly critical parameter to take into account during the biosensor design process. Following our discussions with OzHarvest, creating something disposable but not biodegradable would be irresponsible from an environmental point of view. Recyclability or biodegradability allow for a better reception of our final product, which is a positive for the synthetic biology movement as a whole. Given that one of the driving motives behind our biosensor is to minimise produce wastage, it would be even more redundant to create a wasteful product.
Sensitivity Whilst sensitivity is inherently important in any sensor, talks with Zespri Kiwifruits enlightened us on exactly how important sensitivity is in the case of our biosensor. For kiwifruit in particular, coolstore ethylene levels must be less than 30ppb. Currently all chemical sensing devices with the exception of a gas chromatographer fail to detect ethylene at a concentration less than parts per million. Any device searching to replace traditional gas chromatography must have the capability to detect ethylene levels at this low level, or will be redundant in this context.
Stability As this biosensor relies on the function of genetically engineered cells, immobilising them in a biosensor must allow them to remain stable. The immobilisation structure itself must also not deteriorate over time or due to exposure of unfavourable conditions.
Response Time Biosensors are limited by the amount of time cells take to carry out the necessary processes to express the sensing and detecting proteins. Introducing mutations, or potentially increasing the strength of the promoter can manipulate this.
There are two main methods of measuring produce ripeness. The first is to use gas chromatography to measure the rate of ethylene emanation as a proxy for the stage of ripening. This is highly accurate and highly sensitive, albeit very expensive, non-portable, and complex to use. This involves manually collecting a gas sample of air from inside a controlled produce storage room or shipping container, and loading it into the gas chromatographer for an output reading to be generated. The second method is on the other end of the spectrum in every way. Cheap, inaccurate, time-consuming and cumbersome, our team saw firsthand during our site visit to Fresh Produce Group clusters of workers manually squeezing samples of fruit from each shipping palette to assess their ripeness
This is a skill, which, according to Avocados Australia, is not taught specifically, but gained from years of experience. Neither methods allow for complete assessment of an entire shipment or stock of produce, and neither are elegant or practical. There is a clear need for a more streamlined, convenient, and reliable method of measuring ethylene levels to assess fruit/produce ripeness.
As explored in the Government section of our Human Practices portfolio, there are many regulations that exist both here in Australia and internationally surrounding the application and production of GMOs. Additionally, our Outreach practices also exposed our team to the widespread concerns and apprehension about GMOs and their potential safety issues.
From curious parents accompanying young children at ‘Science in the Swamp’ to retired professionals at the Ku Ring Gai Rotary presentation, the main point of concern derived from the idea of genetically modified living cells being in such close proximity to fresh produce.
Whilst this does seem like a mountainous flaw in the objective of our project, it need not be. Rather, it provides our team with the chance to explore and design some novel, groundbreaking techniques in biosensor design to circumvent this “problem.”
Furthermore, viewing our project in this way neglects the crucial engineering phase in the development of any new technology. E.coli is the host for our biocircuit in the context of the lab, and is essential in proving the fundamental scientific function of the isolated Mycobacterium NBB4 genes EtnR1 and EtnR2. That’s not to say, however, that E.coli must play a part in the transition of this biotechnology from lab to market. One of the biggest potential areas for further research, and what our team envisions for this technology in the future is the development of a cell-free biosensor.
When assessing currently available and emerging technologies, there are many ways to potentially immobilise whole cells such that a functional biosensor can be developed with a much lower, if not completely negligible, biosafety risk. This is a realistic and reasonable phase of development before advancing to a cell-free model.
Polymer anchoring A simple and established method of enclosing cells, enzymes and proteins into sensing devices is through anchoring of these molecules into various polymers. There are many examples of these, including polyacrimide, agar, agarose and chistosan (Close et. al., 2009). Immobilisation occurs due to affinity interactions (Cosnier and Holzinger, 2010), however reaction times are often slow because of diffusion through the polymer (Close et. al., 2009).
Sol-gels Sol-gel is the name given to a class of materials that are formed when a three-dimensional network is formed out of solid particles that are suspended in a liquid as a colloid (Brinker and Scherer, 2013). These gels are most commonly silica-based, and can be described as ‘porous glass gels’ (Close et. al., 2009). Bakul et. al. (1994) presented that silicon sol-gels can effectively trap and immobilize proteins in their matrix with minimal impact on the function, specificity or selectivity of the protein. However, Kuncova et al. (2004) emphasises that the encapsulation process is not optimal for whole cell as the silicon content can result in cell toxicity, while Flinkinger et. al. (2007) attributed increased cell death to alcohol toxicity when lyogels are formed via crosslinkage.
Electrospinning Another novel technique involves spinning fibres of polymers similar to those used in polymer anchoring tainted with cells into a nanofibre matrix (Close et. al., 2009). Stretching individual drops of the polymer cell solution by electrostatic means creates the fibres. Work by Salalha et. al. (2006), found that such method allows cell viability to remain for a three month period when stored below -20 degrees Celsius. Klein et. al. (2009) proved that this technique is successful in cell entrapment for use in bioremediation contexts, despite reduced cell activity.
Hydrogels Many technologies take advantage of the ease of creation of hydrogel structures, such as gel beads, to render cells functionless for use in a biosensor capacity (Ahmed, 2015). As indicated by Flickinger et. al., (2007), the required thickness of these gels creates a high mass transfer resistance. In spite of this, the pores have thin walls and are macroscopic, increasing the risk of contamination incredibly. Despite work by Okamura and Ito (2001) to add crosslinking of molecules to this process, which increased the structural and mechanical integrity of hydrogels (Kopecek, 2007), they cannot be attached to other surfaces as would be required in this context (Flickinger et. al., (2007).
Latex Flickinger et. al. (2007), presented a novel way to immobilize cells via the creation of a nanoporous latex biocoating. The method involves creating a mixture of latex enhanced with carbohydrate complexes and cells (50% v), and rolling it on any surface using a Meyer pull-down rolling system to a thickness of less than 100um. Upon setting, the presence of polymer particles allows for the formation of nanopores that will allow small micromolecules such as ethylene in, but will be too small to allow cells to permeate out. These nanopores also allow for nutrients to reach the cells to increase the shelf life of the coatings. The drying process results in desiccation of the cells such that they can no longer replicate, but can still retain protein/transcribing functions. The cells are thus permanently immobilised in the coating. Such method has a low capital cost, and is very resource efficient.
The paper/acetate: sensing core
By applying this latex nanoporous coating to a cheap, biodegradable, readily available material like paper, or an equivalent cellulose blend, there is scope for various applications. This will be the core technology of all biosensor designs, as the paper can be cut or distributed in many forms. A schematic for this method can be seen below. After GMO cells are grown up, they are mixed with the latex solution and rolled on sheets of paper using a Meyer roller pull-down system. They are then cut into sticker shapes before being coated with adhesive and stuck on produce.
The latex and cell coated paper sensing strips are the core of the final product design. The fundamental strength in this design is the versatility of applications for these strips.
In developing the final biosensor construct, many different things had to be considered. The most influential factor in designing these devices, and the reason for designing multiple devices, was because of feedback from the potential consumers of our product. Experts from Zespri, Avocados Australia, and Fresh Produce Group, all had unique views on how the biotechnology should be manifested into a final design, based on differing needs.
Avocados Australia was optimistic about a sticker design to help consumers select fruit at the ideal stage of ripeness without damaging the fruit through manual handling.
Zespri prefer to control the ripeness of their produce before it hits the shelves of a supermarket, so consumers can purchase fruit of a consistent stage of ripeness and quality. For this reason, Zespri requested a way to monitor ethylene levels in specific shipping crates in an easy and user-friendly way, technology that would eliminate the need to unbag and re-bag thousands of kilos of kiwifruit for the pure purpose of manual inspection.
Fresh Produce Australia implored the importance of manual handling in their section of the processing chain, because it serves the purpose of eliminating bruised or infected pieces of fruit, as well as acting as a ripeness checkpoint. Once fruit has been unpacked from a shipment, it undergoes rigorous quality assurance checks, is ripened to optimal conditions in ethylene ripening rooms, or is stored under refrigeration. The fruit is required to be inspected for size, colour and other physical features as well, all of which cannot be done by technology. For this reason, whilst our technology might be useful for ripeness monitoring, it would not replace the manual handling stage of this process. Instead, FPA suggested that perhaps a sensor that could remain in one of the cold rooms and send a live feed of ethylene levels to a computer in a cost-effective way would be a useful application for their context. For example, FPA handles shipments of kiwifruit arriving in Sydney on behalf of Zespri given they already have the facilities to do so. As part of this arrangement, there are temperature sensors installed in the cold rooms that send a constant temperature reading to Zespri in New Zealand, to ensure FPA are complying with Zespri’s required temperature guidelines. An ethylene sensor could thus serve a similar function.
Consequently, multiple designs were brainstormed and evaluated.
Fruit sticker A small, oval-shaped fruit sticker identical to current ones used to label the origin of all fruit produce could be applied to produce. The latex/cell-coated paper could be coated on the underside with an FDA-approved adhesive. These stickers could be applied to the produce at the same time as other sticker labelling, such that no extra processing would be required.
Scanner A hand-held scanner device, designed for workers responsible for monitoring or handling fruit in the transportation or storage process. A slip of latex/cell-coated paper could be slipped into the front of the device prior to each use, and the device could be pointed at each crate/shipment/piece to measure the cumulative ethylene levels to which it has been exposed.
Curved handheld stick Shaped like a spoon, with a narrow body designed to fit into the palm of the hand of the user and a slightly larger round head with a viewing window at the top, this sensor is also designed for workers monitoring storage rooms or shipments. A latex/cell-coated paper strip is to be inserted into the top of the device for each use, which can be seen through the viewing window.
Industrial sticker A modification of the fruit sticker, this industrial sticker would have a more durable cellulose blend base and would be larger in size. It could also feature shipping information or labelling to reduce the packaging on a crate. This design is ideal for industrial use, such as in shipping containers or on produce storage shelves. It has the potential to give an average reading for a sample of produce, rather than an individual cumulative ethylene reading.
Plate A small, thin square plate designed to sit in the corner of a storage room, this design has a groove for the user to slip a strip of latex/cell-coated paper to allow for ongoing monitoring of an environment. A worker can gain an instant reading periodically by taking a photo of the plate using the app designed for this purpose. A fresh strip can be placed into the plate every hour/day/month depending on the need, and the constant groove ensures a consistent placement of the sensor strip in the room. This eliminates the impact of airflow, ventilation and height, meaning the readings will be comparable.
Swing tag As an alternative design to a sticker, a swing tag could be attached to nets of fresh fruits, such that the customer can gain an average ripeness rating of the bag.
The three most promising designs are the fruit sticker, the plate, and the industrial sticker. The first meets the needs of Avocados Australia; the second could be used by FPA and the third by Zespri. These three products were chosen to be the focus of further product evaluation. Initial designs were sketched and rendered online:
Prototype iGEM Hamburg 2016 have been working with us in many ways, and one of the main points of collaboration was the creation of a prototype of our plate design. Their nanoscientist, Steffen, rendered our plate design online, and created a 3D printed prototype for us!
Given stickers are a less unique design, and are also not allowed at the Hynes Convention Centre, we did not make a prototype.
Areas for Improvement There are some drawbacks associated with this specific design of the technology that should be considered.
Firstly, despite the cells being immobilised in the latex coating, genetically modified E.coli cells are in contact with fresh produce intended for consumption and could potentially be consumed themselves. Due to unease in society regarding GMOs, and also heavy legal restrictions surrounding the release and applications of GMOs, there is anticipated to be a large backlash or consumer concern raised about this issue.
Hopefully outreach events similar to the ones we held, and a growing understanding of synthetic biology will help to diffuse this concern. That being said, regulation of the strength and one-directional permeability of the latex coatings will need to be strict from a biosafety point of view.
Another consideration is the latex allergy. A portion of the population has a non-life threatening allergic reaction to latex. It is unknown whether the quantity or application of latex in this case will trigger such reaction, but it must be noted as a consideration nonetheless.
Interactions The impact this technology will have on other processes is not extreme. The most significant impact that can be expected is a loss of employment. Current industry best practice requires significant labour input to hand check fruit for firmness/ripeness. A capacity to cheaply and accurately use ethyele measurements as a proxy for fruit ripeness would mean that industry would be able to significantly reduce labour requirements. A cheaper, faster and potentially more accurate method for determining the stage of ripening in fruit would also potentially mean that produce imports and exports will increase as the risk associated with produce wastage due to rotting/spoilage/over-ripening during international shipments will decrease. Increased global fruit movements could potentially increase the risks associated with cross-border biocontamination. This could be combatted by strict border quarantine routines.
Another potential disruption comes to mind when assessing the future areas of research that stem from this proposal. Once ethylene measurement has been optimized and made practical, accurate and easy, there is the potential that ethylene manipulation will be more prevalent. That is, an easy measuring system will allow for more accurate and dynamic manipulation of ethylene levels. In general, this would be expected to mean less waste across the perishable fruit and vegetable supply chains. Spoilage leads to losses globally of up to 50% of fresh produce, depending on the type of produce. An ability to cheaply and accurately measure ethylene may help producers, retailers and consumers in developed and developing countries to significantly reduce fruit and vegetable losses.
Positives There are many positive environmental impacts of our biotechnology. From an application point of view, minimizing produce wastage results in less landfill, less garbage, and a higher return on agricultural land. Also, the stickers are 100% biodegradable, meaning that their environmental footprint it extremely low as they will break down completely. Designing the stickers to be biodegradable rather than recyclable is much more practical as consumers are much more likely to dispose of the sticker with their fruit scraps in a normal bin, rather than separating the sticker to dispose of for recycling.
Negatives It can be argued that individual labelling of produce adds unnecessarily to the waste stream. Individually, the stickers would be small, but cumulatively the millions that would potentially be used, even if they are biodegradable, add to the environmental impact of fruit production. This can be weighed against the reduction in food waste that is expected to flow from better control of ripening. However, full life-cycle assessments (LCA) should be carried out on various alternative versions of the product in order to minimize potential environmental costs.
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