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From 2011.igem.org

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As part of our human practices work, we need to consider what will happen in the event that these bacteria are released into the soil. The potential consequences of their release relate to their uncontrolled spread and the possibility that they pass on the auxin genes to a potentially pathogenic bacterium.

To help towards these issues, we have designed a few safety features that will go some of the way towards making this project safer.

Kill Switch Designs

The first idea was to implant a chemical into the selected region of soil that the bacteria would be unable to live without, but this was dismissed on environmental reasons as there is very little that we can add to the soil without damaging its composition or the organisms within.

The second idea was to implement the Holin/Anti-Holin regulated kill switch designed by the Berkeley 2008 iGEM team under the control of a UV sensitive promoter. Since UV light can only penetrate 0.3mm into soil, this would be an ideal method to ensure that the bacteria remain underground.

The third idea uses the same BioBrick parts from the 2008 Berkeley iGEM team to create a toxin/anti-toxin system to try and limit horizontal gene transfer. The Antiholin gene will be on the genome of the bacteria under the control of a strong promoter. The Holin and Lysozyme genes will be present on the same plasmid as the two auxin genes. The idea here is that the presence of the antiholin will prevent the cell from lysing from the effects of holin and lysozyme. In a different cell, i.e., one that does not have antiholin on its genome, the antiholin and lysozyme will kill the cell, preventing it from keeping the plasmid containing the auxin genes.

Cloning Strategy

The first step is to transform competent E.coli cells with the killswitch cassette from Berkeley 08, available in the registry. This will then give us many copies of the genes so that we can use them in transformations.

Next, we will need to obtain a source of the antiholin gene by itself, and this will be achieved using PCR.

The next, and quite possibly the hardest step will be to transform E.coli so that antiholin is present on the genome. For this, we will use versatile conditional-replication, integration, and modular (CRIM) plasmids. The CRIM plasmid is designed so that it integrates into a specific site on the genome, allowing us to ensure that there is only one copy of the gene on the genome.

Promoter Design

The key part of this module is the need for the amount of Antiholin to be equal to the amount of Holin so that the auxin-secreting cells are not lysed. However, the promoter needs to be strong enough to allow enough holin/lysozyme production so that the recipient cell will be lysed quickly.

This is a problem that we hope to resolve through computer modelling, buy building a system that will give information on the required promoter strengths.