Team:Fatih-Medical/Dracoli/Design

Dracoli

• Overview
• Goals
• Design

Design

Monitoring a synthetic compound production system is one of the biggest problems on-going in iGEM. In the parts registry, it is possible to find several inducible promoters which are compatible with various organic and inorganic materials. Yet, the modularity and the ability to be tuned of these parts are limited; for the prospective projects in iGEM, the teams will need to tune the major regulation system in their synthetic devices at the levels of transcription, translation and post-translational modification phases.

This year, we introduce a light tunable synthesis system that includes an engineered BioBrick consisting of two subparts. (Figure 1) Halorhodopsin (originating from Halobacterium) is a light inducible ion channel which is located in the cell membrane and accepts chloride ions into the cell depending on the gradient of ions. (Figure 2 and 3) It is one of the important adaptations for Halobacterium to live under high salinity conditions¹. It was fully characterized by Hong Kong-CHUK 2011 iGEM team last year and was selected as Best New BioBrick, Natural in Asia Regional Jamboree.

The second component of our system is a chloride inducible promoter, Pgad, which is activated at low pH levels that is conducted by chloride ions. (Figure 4) Normally, Pgad operon provides hydrochloric acid feedback mechanism to adjust intracellular metabolism, in order to survive in acidic environment². Pgad has a gene before named gadR, which is constitutively expressed under the control of PgadR. It is a positive regulator of Pgad coupled genes while intracellular chloride level is elevated ².

We presume that after the induction by the emission of the light with the appropriate wavelength, halorhodopsin will become activated and will begin to transfer chloride ions which results high intracellular concentration of chloride and decrease in the pH of bacterium cell. After the transfer of huge amount of chloride ions into the cell, hopefully, our promoter will start to express the downstream genes.

In conclusion, we will hopefully be able to regulate the whole system by changing the parameters related with the light. (Wavelength, power, duration of exposure)

Despite the opportunity of implementing such system in different projects, we chose to use this mechanism to induce self-destruction in E.coli in order to prevent the possible production of genetically modified organisms (GMOs) in the media. With the help of halorhodopsin, we hope to regulate the self-destruction mechanism easily through light parameters. We plan to succeed this by adding different lysis cassettes at the downstream of light-regulated halorhodopsin-Pgad complex.

For the self-destruction mechanism, we intend to use two different devices. Endolysin-holin complex is a commonly used cell killing device by teams in iGEM. The holin perforates the inner cell membrane of the bacterium which ends with several pores on it. (Figure 5) Aftermath, endolysin passes through these pores in order to proceed to the periplasm and cleaves the outer membrane of the bacterium which results the death. (Figure 6 and 7) The second mechanism we plan based on LALF protein. (Limulus anti-lipopolysaccharide factor) It binds strictly to the lipopolysaccharide (LPS) which is the main material of gram negative bacteria cell wall³. Last year, our team showed that LALF can be used as a growth inhibitor for E.coli. Therefore, we aim to use this system to stop all gram negative bacteria in the media to avoid any gene transfer.