Team:Imperial College London/Human Practices/Report

One possible application for this fast response module is in the detection of water-borne parasites. We realised that synthetic biology is rarely used in the fight against neglected tropical diseases (NTDs), and we wanted to be pioneers in this field.

Over 1 billion people - a sixth of the world’s population - suffer from one or more NTDs (WHO, 2007). Despite affecting over 90% of the world’s population in one way or another, only 10% of worldwide health research funding goes into finding preventative or curative treatment for NTDs. This statistic was published by the Global Health Forum and is now referred to as the ‘10/90 gap’ (Global Forum for Health Research, 1999). The 10/90 gap probably exists because of the fact that the majority of people affected by NTDs live in developing countries (Hotez et al, 2006), and so profit margins for NTD drugs are negligible. Therefore, Western pharmaceutical companies are unlikely to see countries in which NTDs are endemic as high priority commercial markets (Hotez et al, 2006).

The World Health Organisation lists the following diseases as the main NTDs (WHO, 2007):

• Buruli ulcer
• Chagas disease
• Dengue/dengue haemorrhaegic fever
• Dracunculiasis (guinea-worm disease)
• Endemic treponematoses
• Human African trypanosomiasis (sleeping sickness)
• Leishmaniasis
• Leprosy
• Lymphatic filariasis (elephantiasis)
• Onchocerciasis
• Schistosomiasis
• Soil-transmitted helminthiasis
• Trachoma

Here's an interview with Professor Alan Fenwick on the subject of NTDs:

NTDs are most commonly found in remote, rural areas of sub-Saharan Africa, Asia and Latin America (Hotez et al, 2006). The effects of NTDs are wide-ranging and include long-term disability, disfigurement, impaired childhood growth, adverse pregnancy outcomes and reduced economic productivity (WHO, 2003). In fact, the effect on worker productivity causes billions of dollars to be lost each year, and retains a country in a state of poverty (Hotez et al, 2006; Molyneux et al, 2005). Added to these problems is that of climate change, which is creating favourable conditions for the spread of many infectious diseases and their vectors, and so it is expected that in the years to come their effects will be felt all the more gravely (Patz et al, 1996).

The construction of man-made water systems has extended snail habitats by creating favourable conditions for their survival and reproduction. As people move closer to lakes, dams and other irrigations systems an optimal environment for the transmission of the parasite results, thus making schistosomiasis even more of a major global health problem (Fenwick, 2009).

In recent years, the treatment of schistosomiasis, has improved markedly due to various schemes of chemotherapy, including the administration of praziquantel (Fenwick, 2009). This drug is effective as a single dose, can be administered by primary healthcare teams and directly affects the Schistosoma parasite (Coon, 2005). However, it should be noted that the primary aim of these schemes is to reduce morbidity rather than prevent transmission.

At present, most of the detection of Schistosoma is only possible after it has infected the human host. There are two main types of diagnosis; antibody detection in blood serum, or egg detection in urine and stool samples (Liu et al, 2010). The latter is more commonly used but it is a lengthy and uneconomical technique. The detection of Schistosoma in the local environment is done rarely, as it is time-consuming and can require cumbersome equipment. Another drawback is the low sensitivity of the tests.

The infective form of Schistosoma, called a cercaria, secretes proteases upon detection of human skin lipids (McKerrow & Salter, 2002). These proteases enable the cercariae to break through the skin barrier and infect the host. We intend to use these proteases to detect the cercariae, because they can trigger a synthetic signaling pathway in our bacterium.

We have discussed a wide range of ethical, legal and social implications (ELSIs) of our project in a Human Practices Workshop, an Interdisciplinary Human Practices Panel Discussion and with [various experts in the field. This has allowed us to ensure that our design specifcaitons are influenced by Human Practices.

As synthetic biology develops as a field, the ease with which systems are put together and the level of automation will improve, and thus entry barriers will diminish. Technology will become more affordable, especially as there is an ‘open source’ ethos within the field of synthetic biology (this will be further discussed in ‘Legal Factors’ and 'Political Factors and Regulation', where we propose an independent, international governing body for synthetic biology).

For more information, please see our Safety Page.

Integration into the B. subtilis genome disrupts essential genes

To make the detection module we will integrate the required genes as two separate pieces of DNA (cassettes) directly into the B. subtilis genome. In order to limit the lifetime of the bacterium in the environment, we will integrate the cassettes at areas in the genome such that they will interfere with essential genes. One of these essential genes is needed to metabolise complex sugars (amyE) and the other is required for pyrimidine biosynthesis (pyrD), which are required for DNA synthesis. Additionally, all lab strains of B. subtilis are tryptophan knock-outs. As a result, the final version of our product would only be able to survive in special conditions where these additional nutrients are provided. Therefore, if it were to be released into the environment, it would, in theory, only survive for a limited period of time (this would be tested experimentally to quantify the exact lifetime of the bacterium in different environments and conditions).

These vectors are now on the Registry and available for use.

Removal of antibiotic genes using Dif excision

Most techniques of introducing new genes into organisms require additional selection genes; these usually confer resistance to a particular antibiotic such as spectinomycin. Our design will also include these genes, however additional Dif sequences built into our design will ensure that these genes are quickly removed from the bacterium, using an inherent control mechanism. As a result, the bacteria used in our final product will not be antibiotic resistant. Integration directly into the genome also ensures that the detection module genes will remain in the organism long enough for the kit to be used effectively.

The product of the output reaction leads to cell death

An additional level of confidence is that a component of the reaction mixture, called catechol, actually kills the bacteria a few hours after it is added. We have tested this and results can be found in Results.

Nevertheless, we are aware that in order for this to occur, the catechol must be added appropriately, and this relies on the correct use of the detection kit. However, if the bacteria were released into the environment without being exposed to catechol, the original genome disruption would be sufficient to ensure that the bacteria would not survive.

We recognise that biological systems are inherently unpredictable and have the capacity to change. Nevertheless, we believe that the benefits of controlling the population and spread of Schistosoma makes this an important project to pursue on the proviso that biosafety issues and environmental impacts are closely monitored, controlled and managed.

We advocate continued, regular testing of the bacteria in order to ensure that any mutations, which would result in the deviation from its function, are noticed early and so the problem can be remediated. We would ensure that there are thorough, controlled and longitudinal studies of the effects of the bacteria on a range of environments and seasons, prior to the marketing of the product.

We wanted to know what control measures were in place at our university and whether or not the government plays a role in this. At Imperial College London, the GM safety committee is responsible for ensuring that all research adheres to the regulations set down by the Health and Safety Executive (HSE). The first stage in the commencement of a project is the completion of a comprehensive risk assessment, which is submitted to the GM safety committee, which will then (i) approve the risk assessment and (ii) identify control measures. These control measures then need to be put in place. If the GM classification is Class 2 or higher, the project must be discussed with the Director of Occupational Health to determine whether health surveillance is necessary.

There has been extensive media coverage concerning access to medicines and technology in the fight against NTDs. In the majority of cases, strong intellectual property rights (IPR) regulations rule out the generic production of drugs so that no alternatives to costly medicines are available. The majority of NTDs occur in developing countries, where healthcare provision is often poor and there are few resources available in terms of NTD medication. Strong IPR regulations for NTD drugs are often seen as unnecessary because pharmaceutical companies will not generate profits because people who can afford the drugs do not need them, and vice versa. Wider access to NTD drugs could be achieved by ensuring that research is available through open source.

The role of industry in synthetic biology is a controversial one. Industry is driven by profit, and we would like to ensure that no company has a monopoly in any particular area through the ownership of one or more of our modules. Ideally, we would like the technology to be available for the manufacturing of generic products.

In particular, universities are centres of learning, and are not profit-generating businesses. We therefore believe that it is essential that research done at universities is accessible and can be used to benefit others. This could be achieved by making the modules available through open source, or by licensing the modules to different companies so that no one company has a monopoly.

• Affordability
• Distribution & ease of transportation
• Shelf life
• Ease of use
• Social acceptability

We also considered the type of organisation who would distribute the detection kits. This could be done by government organisations and local authorities, which would be an ideal solution because it would allow developing countries to organise and manage their local distribution and use of the kits, or use existing infrastructure if it is already in place. However, this may not be the best option if government resources are poor or if there is a threat of corruption or discrimination. If this were the case, aid programs and NGOs may be in a better position to distribute the kits.

We do not believe that our detection kit on its own can solve the problem of Schistosoma. Improved sanitation and education is needed in combination with the environmental control of parasites. It would be necessary to carry out an assessment of the level of education regarding parasitic infections, in particular schistosomiasis, in order to determine where further education was needed. This education programme could be given alongside the training in the use of the detection kit to ensure the long-term feasibility of our project, and also to overcome any cultural boundaries there may be.

It is also essential that we determine the social acceptability of the kits before we finalise the design. During this process, it is possible that we would find that different designs are more appropriate in different settings, and the overall design may be needed to be tailored to a specific community, depending on the setting in which it is implemented.

The benefits to local communities are unlikely to be seen straight away. This is due to the nature of NTDs and their prolonged effects on individuals, and the fact that much of the problem with them is due to socio-economic factors (Hotez et al, 2006; Molyneux et al, 2005). However, as the incidence of, for example, schistosomiasis decreases, the economic productivity of a particular population will hopefully improve, and this should result in a positive feedback loop where the NTD incidence decreases even further due to the improved economy of the given community.

We considered the benefits of targeting the detection kit for use by organisations instead of domestic use. One advantage is that training in the use of the kit would only need to be given to employees of the organisations, and not to whole communities, thus making the system much more viable in terms of cost and time. Also, disposal of the kits after use by individuals was too great an issue in terms of infrastructure. We therefore decided to target it for use by local authorities so that water can be tested in different regions.

Our project involves taking genes from a pathogenic bacterium, Streptococcus pneumoniae, which is by no means ideal. However, an alternative could not be found in a non-pathogenic species. These genes are only involved in the competence pathway and not in any pathways that may confer pathogenicity. There are also homologous genes found in non-pathogenic bacteria. Therefore, we do not expect any adverse consequences from using these genes out of context, and we would ensure that testing is done prior to the marketing of our product so that it is certain that no pathogenicity is transferred to our bacterium.

We chose B. subtilis as our chassis as it is a non-pathogenic bacterium that is present in many environments, such as soil. Using B. subtilis means that the cells can be transported in spore form, allowing them to withstand large temperature extremes. This means that the whole kit will be easy to store and transport, and so will be straightforward to use in resource-poor settings. Before use, the spores would need to be germinated, but this represents no great problem.

Another advantage of using B. subtilis is that we can remove antibiotic resistance genes using Dif sequences, so the final synthetic organism will not be resistant to antibiotics. These antibiotic resistance genes are essential for selection during cloning. During the assembly of our constructs, we used two different antibiotics to select for cells that contained plasmids that contained either the spectinomycin or chloramphenicol resistance genes. Either side of these resistance gene casettes are Dif sites. When the constructs are integrated into the B. subtilis genome, they are relatively stable and cannot be lost, so the resistance genes are no longer needed. At this time, the Dif sites are targeted by an inherent enzyme which all B. subtilis cell possess, namely a recombinase. This recombinase removes the stretch of DNA between each Dif site, including the antibiotic resistance genes.

We considered different types of output, such as an odour or light. However, the most reliable and the fastest way of getting a response appeared to be by using the C2,3O reaction, which has a colour output. Our only concern about this was that when testing water with a high level of sediment, it may be hard to observe the colour change, which is yellow-orange. If this were the case, an odour might be a possible output.

The design of the final product would depend on whether it is more appropriate to test a fixed volume of water using a container, or to use a dipstick to test a variable amount of water. The latter technique would allow one to increase the sensitivity of the test by attracting cercariae using certain lipids, which could be released by the kit. This would lead to the establishment of a concentration gradient of lipids, leading to a higher number of cercaria to be detected.

We intend the test to be quantitative, but this would require a spectrophotometer. However, we have looked at ways to implement quantification by eye, much like a pH indicator test.

Due to the modular design of the detection kit, there are many other applications that would benefit from a fast response module. There are also many other parasites that could potentially be detected, especially those that use proteases to infect their host, such as hookworm, guinea worm and strongyloides. These could be other potential applications of our system.

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Fenwick, A. (2009) Host-parasite relations and implications for control. Advances in Parasitology. 68:247-261

Global Forum for Health Research (1999) 10/90 Report on Health Research 1999

Gryseels, B., Polman, K., Clerinx, J. & Kestens, L. (2006) Human schistosomiasis, Lancet 36, pp. 1106–1118

Hotez, P. J., Ottesen, E., Fenwick, A., & Molyneux, D. H. (2006) The neglected tropical diseases: The ancient afflictions of stigma and poverty and the prospects for their integrated control and elimination. Hot topics in infection and immunity in children III. New York: Kluwer Academic/Plenum Publishers.

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Liu, Z. S., Zhang, L., Yang, H., Zhu, Y. H., Jin, W., Song, Q. A. & Yang, X. L. (2010) Magnetic microbead-based enzyme-linked immunoassay for detection of Schistosoma japonicum antibody in human serum. Analytical Biochemistry 404:127-134

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