"

From 2013.igem.org

Safety

The One Ring to Secrete Them All

The one ring to secrete them all is a customizable secretion pump which uses Escherichia coli nissle 1917, a common probiotic, as the delivery organism. The genes to be expressed and secreted originated from Bacillus subtilis subtilis 168. The organism in which the parts were grown up and assembled is Escherichia coli Dh5 alpha. All of these organisms are rated at a BioSafety Level 1. We have the facilities to handle organisms of BSL 2, but since the organisms we were using were BSL 1; we operated at a BSL 1.

Each of the parts used have their origins in organisms having a BSL 1 or have no rating according to PHE.gov. The plasmid with our system is prevented from being used if transferred to other organisms due to the anti-horizontal mechanisms we have put in place. We use a CymR repressor that binds to the T5-Cumate operator which prevents the Tse2 toxin from being expressed. The purpose of this is so that if any organism were to uptake our system the Tse2 toxin would kill the organism as long as it does not have a gene for the CymR repressor. If there was any reason we would need to remove E. coli nissle 1917, the introduction of P-cumate to the system will prevent the CymR repressor from binding to the T5-Cumate operator which will induce the expression of Tse2 toxin, leading to the death of E. coli nissle 1917. The Tse2 toxin is categorized as BSL 2, but is exempt from the restrictions since it is only an effective toxin when it is inside the cell expressing it. The anti-horizontal transfer method was from parts made by the 2012 Trieste iGEM team. The complete plasmid we are creating secretes cellulase. The anti-horizontal transfer safety mechanism we use has been tested before and found successful. The strain we are using is a well-known probiotic that can be cleared from the user of the organism using p-cumate, if the organism becomes malicious.

Download Safety Form Part 1