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.

Restriction Enzyme System

Aim:

To design a plasmid self-digestion system.

Experimental System:

We are testing different combinations of promoters and restriction enzymes. We have to characterize both the promoters (by measuring the expression of RFP) and the restriction enzymes (by measuring killed cells).

Achievements :

• Construction of 4 biobricks [Read more]:
  • [http://partsregistry.org/Part:BBa_K914003 K914003]: L-rhamnose-inducible promoter
  • [http://partsregistry.org/Part:BBa_K914005 K914005]: Meganuclease I-SceI controlled by pLac
  • [http://partsregistry.org/Part:BBa_K914007 K914007]: Meganuclease I-SceI controlled by pBad
  • [http://partsregistry.org/Part:BBa_K914008 K914008]: Meganuclease I-SceI controlled by pRha
• Demonstration that all 3 generators (K914005, K914007, K914008) work and express I-SceI meganuclease in cells. [Read more]

• Characterization of 2 biobricks from TUDelft [Read more]:
  • [http://partsregistry.org/Part:BBa_K175041 K175041]: p(LacI) controlled I-SceI homing endonuclease generator
  • [http://partsregistry.org/Part:BBa_K175027 K175027]: I-SceI restriction site
• Characterization of the L-rhamnose-inducible promoter (pRha).

MAGE

Aims :

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 Aim :

Creation of a novel protein import mechanism in bacteria.

Experimental System

Exploit the natural Colicin import domain fused to any protein at will, dubbed here: "Synthetic Import Domain".

Achievements

• 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

Aim : Harness bacteria-containing gel beads to assure cell containment and complement activity of genetic safety systems.

Experimental system: Bacterial cells are encapsulated in alginate beads. We used a cell containment assay based on plating to assess the release of cells from alginate beads. In addition, we aimed at improving the entrapment of cells through stabilization by polyethyleneimine and covalent cross-linkage by glutaraldehyde.

Achievements :

• Encapsulated cells achieved and their ability to propagate and express proteins within alginate beads demonstrated.
• Stabilized alginate beads by covalent cross-linkage achieved and their ability to entrap cells demonstrated.
• we performed additional characterization of the Bristol 2010 nitrate reporter [http://partsregistry.org/Part:BBa_K381001 K381001]
• Efficient killing by colicin producing cells was achieved within the beads.

test

Human Practice