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From 2011.igem.org

Human Practices

Containment

To prevent spread of the auxin-producing plasmid in the environment, we have devised a containment device that will be able to kill other bacteria that take up the plasmid. In addition, we have devised experiments to test the survivability of E. coli in soil to evaluate whether these bacteria would be outcompeted by other soil microorganisms. Soil-dwelling protozoa also play an important role as they have been shown to feed off bacteria.

Soil Experiment

This experiment is designed to determine the survivability of E. coli in soil. If bacteria were to be left in the soil we can estimate accurately the length of time they will be alive by carrying out this experiment. It is probable that the bacteria will live for longer in sterile soil than non-sterile soil, due to factors such as competition or they are being attacked by soil bacteria. This experiment was started by our work experience student Kiran and carried on by us after he left.

For this experiment, we placed soaked small discs of filter paper in GFP expressing E. coli and put these into autoclaved and non-autoclaved soil. The bacteria were incubated for 10 days. Cultures were grown up in ampicillin and/or kanamycin containing medium every day to measure the optical density and thus determine how many bacteria had survived in sterile and non-sterile soil for set periods of time.

To ensure that we were not growing up antibiotic-resistant soil bacteria, we grew up cultures from non-inoculated soil as negative controls. In addition, we grew up cultures on ampicillin and kanamycin containing plates on day 6 to check for fluorescence. There were no colonies detected on the negative control plates, showing that OD measurements in the negative control were due to soil particles in the media. The colonies grown up from E coli inoculated in soil were brightly fluorescent, confirming the presence of plasmid-carrying E coli in the soil after a long period of time.

Subsequently, we stopped measuring the OD of bacteria and started plating out cultures from sterile and non-sterile media to check for presence of the plasmid.

Results

Non-Sterile:

1A 0.315
1B 0.393
1C 0.361
2A 0.553
2B 0.583
2C 0.548
3A 0.399
3B 0.668
3C 0.300

Sterile

1A O.523
1B 0.548
1C 0.476
2A 0.716
2B 0.616
2C 0.664
3A 0.950
3B 0.887
3C 1.002

Containment Device

The containment device is based on a toxin/anti-toxin system taken from the lysis cassette made by the Berkeley 2008 team. This originally involved the secretion of holin along with lysozyme, so that the holin would form pores in the inner membrane and allow the lysozyme to break down the cell wall. This will cause the cell to lyse when the inducible promoter was induced by arabinose.

Also included in this cassette is the gene for antiholin, under the control of a weak, constitutive promoter to prevent any leakage.

Our project takes this a step further. By using the anti-holin's ability to inhibit the activity of holin, we can create a system in which the lytic activity of holin can be negated by the presence of antiholin in certain cells. This means that we can make a plasmid that can kill one cell, but replicate in another.

Panel

As part of our human practices process, we held two panels. The first panel consisted of Prof Richard Kitney, Dr Tom Ellis, Dr Guy-Bart Stan, Charlotte Jarvis and Kirsten Jensen.

The panel addressed many different questions that we later used to inform our design.

Could the bacteria impact the germination of the seeds?
The coat itself would not be prohibiting germination. It is possible to design the coat sufficiently well to ensure that this would not happen. In addition, seeds normally germinate in soil full of bacteria that do not prevent germination.

How can we ensure that the auxin does not kill the plants?
We will be able to vary the inoculum of bacteria in the coat. We will get an experimental estimate of the auxin production, which will help us estimate the ideal number of bacteria to be contained in the seed coat. While a weak promoter may be better for constitutive expression of auxin, it will be easier to weaken the promoter later. We have used an insulator sequence to separate the promoter from the RBS so that we will be able to replace the promoter very easily. This may also contribute to the fine-tuning of expression and thus help us make sure that auxin is expressed at ideal concentrations. The worst case scenario consists of the auxin producing genes being transferred to other bacteria that become pathogenic. However, this could be tested exclusively beforehand and the infrastructure for this separate development and safety testing stage is already in place. In addition, unlike synthetic auxin, natural auxins such as IAA have a short half-life and degrade rapidly.

What is the risk-benefit relationship of our implementation?
In our implementation, we are trying to improve already existing practices. We do have to take a certain risk to combat desertification. However, is putting GM bacteria into soil worth speeding up the acacia tree planting process? How much does this really help? While GM bacteria may pose a risk, introducing foreign plant species that also show drought resistance and grow faster than acacia trees can be extremely risky and introduction of foreign species into ecosystems has already had negative consequences all over the world. This effect is likely to be worsened by the fact that we would be introducing the foreign species into an already damaged ecosystem. We may also be able to plant other fast growing plants at the same time as planting our coated seeds to hold the soil down while the seeds are growing.

Should we be using B. subtilis or E. coli as our chassis?
B. subtilis spores spread very easily over long distances and may thus be blown into different ecosystems where they may have negative effects. On the other hand, its spores would be easier to integrate into a seed coat. E. coli is not as easy to integrate into the seed coat. However, it does not form spores and is therefore very likely to stay inside the ecosystem we introduce the microbes into. We have already shown that E. coli is able to survive in non-sterile soil for more than two weeks and that it can pass on its plasmid to other bacteria, enabling them to express GFP and antibiotic resistance. By using E. coli as our chassis, we can be sure that the bacteria will survive in the soil for a reasonably long period of time but not spread as rapidly as B. subtilis spores would. At the same time, we will be preventing plasmid transfer using the BacTrap. We should be able to overcome the technical challenge of putting E. coli into the seed coat.

Would we be able to get rid of the bacteria once they are in the soil?
The kill switch is never 100% effective and the bacteria will lose the plasmid. In addition, bacteria killed by kill switches still leave behind DNA that can be conjugated by other, naturally occurring bacteria. We may not be able to take the bacteria back out of the environment after they have been distributed into soil. Instead, we will be aiming to prevent spread of the plasmid. We will be using E. coli as our chassis, which should be outcompeted in the soil and our BacTrap device will be used to prevent plasmid conjugation.