From 2011.igem.org

Revision as of 18:52, 28 October 2011

Informing Design

We consulted numerous experts in various fields to ensure that the design of the AuxIn system respects all relevant social, ethical and legal issues. One module of our system, Gene Guard, is a direct result of brainstorming around the issues involved in the release of genetically modified organisms (GMOs). Although we have only reached the proof of concept stage, we have put a lot of thought into how AuxIn may be implemented as a product and the legal issues that would be involved.

Ecology

Since our project involves releasing bacteria into nature, we have to consider their impact on the environment and other soil organisms. This is vital in assessing the safety of release. We did a literature review of the impact of natural auxins on the rhizosphere and consulted ecologists to find out more about the impact our GMO could have.

The basis of our project - plant-growth-promoting bacteria

The fact that there is a close mutualistic relationship between plants and many species fungi such as micorrhiza has been known for a long time. However, plants also live in close symbiosis with bacteria. Much like the human body, a plant interacts with bacteria living in proximity to, on the surface of, and inside the plant itself. Symbiotic bacteria benefit plants in many ways. They help fend off parasites, take up minerals from the soil, enhance stress tolerance^[7] and increase growth^[8]. Many bacteria produce chemicals to stimulate plants, one of which is indole-3-acetic acid, a type of auxin ^[9].

Our project taps into this naturally existing symbiosis to engineer plants using a bacterial delivery system of compounds. Using the tools of synthetic biology, this root growth promoting mechanism has been engineered in order to combat desertification.

There are many advantages of using this approach; the most straightforward one is the fact that engineering bacteria is easier than engineering plants. This also makes it possible to have tight control and fine tune how the bacteria carry out their task.

In addition, some plants are very difficult or even impossible to engineer genetically and using a bacterial delivery system can help overcome this difficulty. Both E. coli and baker's yeast have been shown to interact with more than just one plant species and it is reasonable to assume that microbes could be engineered for optimum plant interaction and compound delivery. When considering a world-wide problem such as desertification that would require a lot of different plant species to be engineered, this simplification of the engineering process harbours many advantages.

Although it can be argued that the improvement of drought-tolerant crop plants via genetic engineering seems promising and there has been great progress made in this field^[10], agricultural environments are much less complicated than natural ecosystems and crop plants are much better studied.

In addition, during our research into the human practices issues surrounding our project, we were advised by several experts that it is unwise to introduce foreign plant species into new areas to combat desertification. It is of paramount importance to use native plant species for our reforestation efforts and therefore the microbe-plant interaction-based delivery system is a very useful approach.

Effect on soil fauna

Figure 1. Dr Alex Milcu demonstrating the Ecotron experimental setup (photo courtesy of Dr Milcu).

1. Goal

Many animals above and below ground are extremely important to existing ecosystems. We want to ensure that there is no significantly detrimental effect on these animals caused by our bacteria.

2. Action

We met Dr Alexandru Milcu, an expert in above/below ground interactions at the Imperial College Silwood Park campus to discuss the impact of our project on the soil ecosystem (see Figure 1).

Dr Milcu informed us that there is no known negative effect of auxin on soil fauna. A more fibrous root network should not influence metazoans to a noticeable extent. However, earthworms take up nutrients through their coelom and may be affected by heightened auxin concentrations, although this has not been established in the literature. Earthworms act as "ecosystem engineers" and are therefore extremely important to the soil. Affecting them negatively should definitely be avoided (Dr Alexandru Milcu, oral communication).

3. Result

We devised a development plan (see the Implementation page) that our project will undergo before release would become feasible. This plan includes extensive testing of the impact of AuxIn on several important organisms, including earthworms, fish and other animals.

Effect of protozoans on our project

1. Goal

An issue with dispersing bacteria in the soil is that they may be eaten by protozoa. Grazing by protozoa has been shown to increase plant growth ^[6]. There are competing theories that this may be due to promoting auxin-producing bacteria or by increasing the nitrogen in the soil. However, it is becoming more likely that the latter theory is true (Dr Alexandru Milcu, oral communication).

2. Action

We met with Dr Milcu to discuss the impact protozoans may have on the implementation of our project. To test the impact of protozoan grazing on our bacteria, Dr Milcu suggested ensuring that protozoa be in test compost with our bacteria and then measuring auxin levels. This would add to the complexity of trials but give a more realistic measure of density and auxin secretion than by simply plating out our bacteria on agar.

3. Result

We unfortunately did not have time to test our auxin-secreting bacteria in compost. However, we have incorporated this experiment into our future implementation plan.

Effect on plant population composition

1. Goal

Monocots such as grasses typically grow fibrous root networks, while dicots tend to grow long, deep branched roots. Auxin influences dicots and monocots differently. While it induces lateral root growth and inhibits deep root growth in both types of plants, only dicots are influenced negatively by exogenous auxin^[3]. Supplying auxin to the soil may therefore result in selecting against dicots and causing a predominantly monocot population. While this may be detrimental for crop usage, dense networks of roots lead to an exponential decrease in water erosion^[4]. Fibrous root networks are therefore advantageous when trying to prevent soil erosion. We want to ensure that our bacteria do not have a detrimental effect on plant population composition.

2. Action

Figure 2. Syngenta visit: Prof. Stuart Dunbar showed us the greenhouses. (Picture by Imperial College London iGEM team 2011).

We met with soil and auxin specialists at Syngenta to discuss the implications of our project (see Figure 2). Dr Torquil Fraser and Dr. John Paul Evans advised us that there are four main natural and four synthetic auxins. Synthetic auxins are very stable and persist for weeks to months in the soil and are commonly used as herbicides. Unlike synthetic auxins, natural auxins are rapidly degraded. In plants, the auxin concentration is tightly regulated by conjugation with aspartic acid and metabolic feedback loop. This is not the case with synthetic auxins because of a slight change in their molecular structure.

In addition, we discussed the issue of skewing the plant population with Dr. Milcu.

3. Result

Skewing the plant population may have negative impacts from an ecological perspective, affecting diversity, but this effect may be counterbalanced by the conservation of plant habitation in general. In addition, we are considering putting our bacteria into soil alongside plants that are able to revegetate desertified areas.

One of the great advantages of synthetic biology is the degree of control we can have on a particular system. We have introduced a sequence between RBS and promoter that facilitates exchanging promoters without affecting the coding sequence. It will therefore be possible to change the amount of IAA secreted by our bacteria to ensure that the amount produced is not inhibitory.

To test the effects of heightened auxin on different kinds of plants, Dr Milcu suggested setting up lab experiments with different plant functional groups. These would be planted in compost with an increased auxin concentration (using synthetic auxin) and observe how they respond. This experiment would be performed in the later testing stages of the project when we would be testing the impact of auxin on the environment.

References and Bibliography

[1] UNCCD (2011) Desertification: a visual synthesis. (Online) Available from: http://www.unccd.int/knowledge/docs/Desertification-EN.pdf (Accessed on 12 August, 2011).

[2] Geist H and Lambin E (2004) Dynamic causal patterns of desertification. BioScience 54: 817-829.

[3] McSteen P (2010) Auxin and monocot development. Cold Spring Harb Perspect Biol, a001479.

[4] Gyssel G and Poesen J (2003) The importance of plant root characteristics in controlling concentrated flow erosion rates. Earth surface processes and landforms 28: 371-384.

[5] Spaepe S et al. (2007) Indole-3-acetic acid in microbial and microorganism-plant signalling. FEMS Microbiol Rev 31: 425-448.

[6] Krome K et al. (2009) Soil bacteria and protozoa affect branching via effects on the auxin and cytokinin balance in plants. Plant Soil 328: 191-201.

[7] Mei C, Flinn BS (2010) The use of beneficial microbial endophytes for plant biomass and stress tolerance improvement. Recent patents on biotechnology 4(1): 81-95.

[8] Sturz AV, Christie BR, Nowak J. Bacterial endophytes: Potential role in developing sustainable systems of crop production. Critical Reviews in Plant Sciences 2000 2000;19(1): pp. 1-30.

[9] Spaepen S, Vanderleyden J, Remans R (2007) Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS microbiology reviews 31(4): 425-448.

[10] Yang S, Vanderbeld B, Wan J, Huang Y. (2010) Narrowing down the targets: towards successful genetic engineering of drought-tolerant crops. Molecular Plant 3(3): 469-490.

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