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 :
  • [http://partsregistry.org/Part:BBa_K914000 K914000] : P_Lac-supD-T : tRNA amber suppressor
  • [http://partsregistry.org/Part:BBa_K914009 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 : [http://partsregistry.org/Biosafety 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).

Delay System

Aim : A programmed delay will allow the cell to perform its intended function before our DNA-degrading suicide machinery is expressed.

Experimental system: We used two different approaches to create this delay. The first one is based on the gradual dilution of a regulatory transcription factor. The second one makes use of a stationary-phase specific promoter. Both systems eventually result in the expression of the restriction enzyme I-SceI. In the final design, I-SceI cleaves the antitoxin gene, ultimately dooming the cell. Each step in this causal sequence contributes to the overall delay in the system.

Achievements :

• Construction and characterization of the dilution delay system
  • [http://partsregistry.org/Part:BBa_K914004 K914004] : P_BAD-AraC-RBS-LacI ; characterization
• Characterization of the sRNA repression system of Yokobayashi et al.
• Cloning of the yiaGp stationary phase promoter

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

Encapsulation

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.

Aim

To chart new venues of best practice for synthetic biology. To this end, we examined the ethical, biological and social concerns related to the release of genetically modified bacteria in the wild.

Metodology

1. Interviews with experts which enabled us to have a broad overview of the state of the art. Read More
2. Interaction with high-schoolers to have first-hand appreciation of reactions from first exposure to synthetic biology
3. We screened previous iGEM team’s wikis to trace the evolution of biosafety concerns and devices in the iGEM community, focusing on proposed containment systems. Read More
4. We focused on horizontal gene transfer as main generic risk factor.
5. Synthetic report where we addressed the concerns raised by synthetic biology per se, that is, as a technique. Then, we analyzed the specific concerns that arise from synthetic biology’s potential applications in nature. [https://2012.igem.org/Team:Paris_Bettencou