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From 2013.igem.org

Overview

We are utilizing the pOSIP plasmid to chromosomally integrate transformed genes into NEB 10 Beta cells and then remove antibiotic resistance genes to prevent horizontal gene transfer of LLO or any antibiotic resistance genes. We are also developing a pH-based gene switch to create a secondary safety mechanism so that LLO is only translated under acidic conditions and translation is repressed by neutral and basic conditions.

pOSIP

pOSIP is a system used to directly integrate genes into the genome of an organism. The ASU iGEM team plans to use this vector to deliver GFP and LLO directly into the genome to create a modular platform for cancer vaccine delivery, which can be altered simply by switching out a plasmid containing the cancer antigen of interest. The PE-FLP plasmid alows for easy excision of antibiotic-resistance genes, at 37 degrees Celsius, that were integrated into the genome via pOSIP, which can be used to develop a non-antibiotic resistant cancer vaccine chassis. Any unintended use of the vaccine could then be neutralized by an antibiotic agent.

PE-FLP expression and removal of antibiotic resistance at 30C v. 37C.

Future Research: LLO pH Switch

The pH Switch depends on a acidic PH-active promoter that acitvated transcription of LLO and another promoter on the same vector that was activated under neutral and basic pH. The neutral and basic pH-activated promoter transcribed an mRNA strand complementary to the mRNA sequence of the RBS downstream of the low pH-activated promoter. The complementary mRNA binds to the RBS mRNA sequence and prevents transcription of LLO, providing two steps of translational regulation.