Team:Paris Bettencourt/Achievements

Semantic containment

Aims

Creating a semantic containment system to prevent gene expression in natural organisms Characterize the system Use this system in all genes of the system, the critical genes first (e.g. colicin) System

An amber codon (stop codon) embedded in protein genes to prevent their expression and an amber suppressor system in our genetically engineered bacteria

Achievements :

Construction and characterization of 2 biobricks : K914000 : PLac-supD-T : tRNA amber suppressor K914009 : P1003* Ser133->Amber Codon : kanamycin gene resistance with 1 amber mutation Both part were well characterized and works well. For the second parts, we show that as expected, one mutation is quite leaky, although it works qualitatively, but one mutation is not enough if we want to release such parts in nature. Other reasons emphasize this observation, notably the weakness of being at one mutation to recover the protein functionality.

Creation of a new category in the part registry : Semantic containment. The aim of this category is to let people improving each part by adding for instance other amber mutations to existing part to increase the containment.

Suicide system

Aims : Implement a kill-switch that features population-level suicide and complete genome degradation.

System : A synthetic toxin-anti-toxin system based on the wild type Colicin E2 operon.

Achievements : We showed that Colicin E2 cells induce cell death in sensitive populations, and that these sensitive populations can be protected by providing them with our engineered immunity protein.

• Construction of 2 biobricks :
  • [http://partsregistry.org/Part:BBa_K914001 K914001] : pLac-repressilator RBS-Colicin E2 immunity protein
  • [http://partsregistry.org/Part:BBa_K914002 K914002] :repressilator RBS-Colicin E2 immunity protein

Part K914001 is well characterized and provides immunity to sensitive cells against the Colicin E2 activity protein, but is leaky. Part K914002 is promoterless and allows users to easily plug in the appropriate promoter for their desired purpose.

• Creation of a new category in the part registry : [http://partsregistry.org/Biosafety XNase]. The aim of this category is to provide users with DNase/RNase parts that can be used for improved kill switches featuring the degradation of genomic material.

MAGE Aim

Removal of four FseI restriction sites from E. coli MG1655 genome.

Experimental System

Using multiplex automated genome engineering (MAGE) - a technique capable of editing the genome by making small changes in existing genomic sequences.

Achievements

Proof of concept by introducing a stop codon in the middle of the lacZ gene

Synthetic Import Domain

• Construction of colicin-like toxin by fusing Colicin E2 based "Synthetic Import Domain" with RNAse domain of colicin D
• Constructon of FseI, I-SceI, LuxR active fragment, LacZ alpha fragment, PyrF and T7 RNA polymerase fused to the two types of "Synthetic Import Domains" from Colicin E2 and Colicin D
• Proof of concept with LacZ alpha fragment fused to "Synthetic Import Domain" from Colicin D

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