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.

Aims

Human concerns arose organically during the construction of the bWARE containment system, and human practices were intrinsic to every stage of our project. In designing and building our best genetic containment system, we often encountered limits on the ability of science alone to measure our performance. When is a biosafety system safe enough? The answer to this question is partially scientific, to the extent that horizontal gene transfer events can be observed and modeled. But the answer is also social, because ultimately the public will decide if a biosafety system works well enough to use. The only way for us to know if bWARE is a success is in conversation with experts and the community.

We propose and implement new ways for iGEM to organize and present biosafety information, both for scientists and the public. We believe our reforms to the BioBrick registry will help synthetic biologists to find the best biosafety tools for their application. We also imagine the beginnings of a quantitative, context-specific biosafety database serving citizen scientists. Practical safety data will feed an informed public forum.

Methodology

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 the main generic risk factor.
5. Comprehensive 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. Read More

Main Conclusions

1. Societal interaction:

   • The need to raise awareness of synthetic biology in the population so people can decide in the most enlightened way possible what they want from this new technology and of its applications (A),
   • The need of a discussion between society’s different protagonists to set goals, define what they would consider as benefits and acceptable risks (B),

2. Best research practice:

   • Zero risk is impossible to achieve as no containment system can be 100% safe (bacteria can always escape by mutations) (C),
   • There is a lack of quantitative data evaluating the probability of failure of any synthetic biology engineered system, in particular containment systems (D),
   • There is a lack of quantitative data evaluating the risk of HGT assuming containment systems failed (E),
   • The compiling of the wiki screen shows that no containment systems created in iGEM is robust: they lack the above quantification and are mostly one mutation away from failure. We call for major effort of the iGEM community to quantify available containment systems and search for new solutions (F),
   • The need for an INDEPENDENT cohort of scientists to test experimentally any application of synthetic biology that requires releasing in the environment (G),

You can find the full list of conclusions here

Main Proposals

1. Societal interaction:

   • Organizing a workshop on synthetic biology and a tour of our lab for 60 high school students, (addresses issue A and B) Read More. First initiative for teaching synthetic biology in French high-school leading to a high-school iGEM team. Ultimately, we would like interaction with high school or middle school students to be a requirement for an iGEM gold medal.
   • Organizing a debate with 10 non expert students from various background, and then opening the debate to the floor (the public), which was made up of both experts and non experts, (addresses issue A and B) Read More.
   • Creating a page to explain horizontal gene transfer to non scientists. Go to HGT page

2. Best research practice:

   • Creating a system as robust as possible, that is many mutations away from failure (this is what our bench work has been all about) (addresses issue C and F),
   • Creating a safety page on the biobrick registry where all the safety devices that exist are listed and characterized (included evaluation of their robustness) in order for iGEM teams to pick the most appropriate device to add to their newly created genetic circuit. Ultimately, we would like the integration of safety modules and risks assessments to be part of of every synthetic biology project from the very start (already listed in the safety page or created de novo by the team) (addresses issue D, F), [http://partsregistry.org/Biosafety Go to safety page]
   • The community has to build a collection of bio-safety devices for future engineers
   • Each synthetic biology application should assess and disclose a list of application-specific risks and hazards.
   • Development and adoption of a safety chasis for synthetic biology research and prototyping.

You can find the full list of proposals here