Team:Paris Bettencourt/Achievements

Semantic containment

• 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

The first part (supD) is well characterized and works well. For the second parts, it turns out that this mutation is quite leaky, although it works in lab conditions, 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.

Resctriction enzyme system

• 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
• Currently we are in process of L-rhamnose-inducible promoter (pRha) caracterisation. [Read more]

Suicide system

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

• Proof of concept by introducing a stop codon in the middle of the lacZ gene using multiplex automated genome engineering (MAGE)

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

test