From 2011.igem.org

Latest revision as of 09:26, 15 September 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

As 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 our organism’s impact.

Effect on soil fauna

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

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).

Effect on soil microorganisms

E. coli do not naturally occur in the soil, which makes it hard to predict its influence on other soil microorganisms. In addition, they are very likely to be outcompeted.

If we want to aim for endurance of our bacteria in the soil rather than ensuring as much containment as possible, using E coli as a chassis may not be viable. Accordingly, we have codon optimised all of our new BioBrick constructs for E coli as well as B subtilis. For future applications, it may therefore be useful to sample soil in the area and determine the dominant bacterial species. Methods for this have already been established and include shot-gun sequencing of all soil microorganisms (Dr Robert Griffiths, oral communication). These bacteria are very likely to persist in the soil and may be used as more applicable chassis.

Another aspect we need to consider is protozoan grazing on our bacteria.

Effect on plant population composition

Monocots such as grasses typically grow fibrous root networks while dicots tend to grow long, deep roots, from which other roots branch outwards. Auxin influences dicots and monocots differently. While it induces lateral root growth and inhibits deep root growth in both types of plants, only diots are influenced negatively by exogenous auxin, on which it can act as a herbicide (McSteen, 2010). Supplying auxin to the soil may therefore result in selecting against dicots and causing a predominantly monocot population to grow fibrous networks of plants.

While this may be detrimental for crop usage, dense networks of roots lead to an exponential water erosion decrease (Gyssels & Poesen, 2003). Fibrous root networks are therefore advantageous when trying to prevent soil erosion.

Auxin-producing bacteria can also parasitise plants and inhibit their growht. Several measures can be taken to ensure that our bacteria are beneficial. Inhibitory effects of auxin can be prevented by producing less than 106 CFU per mL and although it seems that pathogenic bacteria are more likely to invade the plants, beneficial bacteria can be found inside of plants as well. (Spaepen et al., 2007).

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. Rather than putting our bacteria into soil and hoping for the best, we are planning to using existing programmes to „reclaim“ deserts and use our bacteria to enhance the growth of these plants.

References:
UNCCD (2011) Desertification: a visual synthesis. (Online) Available from: http://www.unccd.int/knowledge/docs/Desertification-EN.pdf (Accessed on 12 August, 2011).
Geist, H. & Lambin, E. (2004) Dynamic causal patterns of desertification. BioScience 54:817-829.
McSteen, P. (2010) Auxin and monocot development. Cold Spring Harb Perspect Biol 2010;2:a001479.
Gyssels, G. & Poesen, J. (2003) The importance of plant root characteristics in controlling concentrated flow erosion rates. Earth Surface Processes and Landforms 28:371-384.
Spaepen, S. et al. (2007) Indole-3-acetic acid in microbial and microorganism-plant signalling. FEMS Microbiol Rev 31:425-448.
Zwahlen, C. et al. (2003) Effects of transgenic Bt corn litter on the earthworm Lumbricus terrestris. Molecular Ecology 12:1077-1086.
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.

Interview with Dr Alexandru Milcu

Dr Alexandru Milcu is an expert in above-below ground interactions who works at Imperial’s Silwood Park campus.He is a research associate and leads the research conducted in the Ecotron facility. He kindly agreed to meet Nick and me at Silwood to discuss the ecological implications of our project. We discussed some of our ideas about inoculating the bacteria into the soil and re-establishing vegetation in areas affected by desertification. He gave us a lot of very valuable advice and pointed out strenghts and weaknesses of the project.

Dr Alex Milcu demonstrating the Ecotron experimental set up (picture courtesy of Dr Milcu).

We discussed our idea of using a capsule to place our bacteria into the soil. Dr Milcu thought this idea was feasible and suggested making the capsule water soluble as one of the main limiting factor for bacterial growth is soil humidity. Another option we discussed was to inoculate the bacteria after rainfall.

An issue with dispersing bacteria in the soil is that they may be eaten by protozoa that naturally occur in the soil. Most bacteria are probably digested in this way. Grazing by protozoa has been shown to to increase plant growth. 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. 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 adds to the complexity of trials but gives a more realistic measure of density and auxin secretion than by simply plating out our bacteria on agar. Realistic conditions are also important for plant experiments.

Dr Milcu thought it was a good idea to plant trees at the same time as putting our bacteria into the soil. He also confirmed our theory that the project would not be very helpful with agriculture, especially because agricultural plants would be uprooted again. In addition, heightened root growth may lead to decreased growth above ground, which would be problematic for crops but should not have a lot of negative consequences for trees. He recommended using a mixture of different plants to re-establish a diverse ecosystem. However, this may be problematic if we would later skew the population if auxin is proven to differentiallty affect different plant functional groups.

Altering the plant community compositon a problem on its own. We do not want to further endanger threatened species or negatively affect diversity. However, it can be argued that we would be targeting disturbed areas with very little naturally occurring diversity. Nevertheless, we would have to ensure containment because we would not want the bacteria to spread to other, more diversely populated areas and alter native plant communities. While our kill switch would be preventing spread, it would not ensure complete containment. To address this issue, we are planning to model the spread of bacteria.

In addition, auxin concentration is a big concern. It should not give the bacteria an invasive advantage or act as a herbicide (although auxin as a herbicide is normally sprayed on leaves). It is therefore adamant that we not only model auxin secretion by our bacteria but also test it.

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 may give us a better idea of how different plant functional groups are affected by auxin.

An issue we need to consider is that E. coli may not be able to survive in soil. This issue is complicated as we want the bacteria to have a lasting beneficial effect on the plant population by secreting auxin. However, at the same time, we do not want the bacteria to spread to other areas and affect diversity or crop yield. Dr Milcu thinks it is a sensible idea to use naturally dominant beneficial soil bacteria to ensure that they will last in the soil. However, these bacteria would not be outcompeted and may therefore be more likely to spread to other areas.

While we will be aiming for uptake of the bacteria into plant roots, Dr Milcu pointed out that auxin will mainly act in the rhizosphere and the bacteria will not have to invade the roots for plant growth to be stimulated. Dr Milcu thought the idea of controlling root morphology by controlling the distance of bacteria from the root is an interesting concept but unlikely to be feasible.

It is currenlty not known how auxin affects soil fauna. Auxin should not affect fauna dramatically. However, invertebrates with cutaneous respiration such as earthworms may be negativelyaffected. Earthworms are very important for soil structure and are often described as “ecosystem engineers“. While the areas that we will be targeting may not have earthworms, we do not want to negatively affect these important soil engineers. Below-ground invertebrates are responsible for nutrient cycling. This may be slowed down if the organisms are negatively affected. To test the effect of auxin on earthworms, Dr Milcu suggested keeping a couple of earthworm species in compost with a heightened auxin concentration, ideally equal to that which we are expecting our bacteria to produce, and observe the effect of auxin on the worms.

Another thing we will need to consider is the impact of our project on the carbon budget. Increased root biomass leads to more carbon storage so that we will probably create a bigger carbon sink. However, we will need to do more research into this.

Dr Milcu also suggested to conduct split root experiments to observe the effect of auxin treatment. In this type of experiment, the root of a plant is split in two and two different treatments are applied to both parts of the root.

In summary, Dr Milcu endorsed our idea of putting plants into the ground alongside our bacteria. He did not think there would be a lot of negative effects on soil fauna and that our project may positively affect the carbon budget. We will need to weigh out if we want the bacteria to persist in the soil against containment issues associated with this. The three big concerns Dr Milcu named were containment, the effect of auxin on soil invertebrates and changing the compositon of plant communities. We will be addressing these issues as well as possible by conducting further research and experiments.

Correspondence with Dr Robert Griffiths