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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.