Team:Paris Bettencourt/Achievements

Achievements :

Semantic containment

• Construction and characterization of 2 biobricks :
  • K914000 : P_Lac-supD-T : tRNA amber suppressor
  • 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 : 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]:
  • K914003: L-rhamnose-inducible promoter
  • K914005: Meganuclease I-SceI controlled by pLac
  • K914007: Meganuclease I-SceI controlled by pBad
  • 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]:
  • K175041: p(LacI) controlled I-SceI homing endonuclease generator
  • 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 :
  • K914001 : pLac-repressilator RBS-Colicin E2 immunity protein
  • 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 : 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

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