Composite parts

New parts submitted to the registry:

Two parts ( BBa_K3373012 and BBa_K3373014 ) were created to be used to construct the engineered strains that we designed for our project. Unfortunately, due to the public health crisis and the experimental constraints that we encountered, only BBa_K3373012 could be synthesized in the laboratory and no transformation in the final host C. reinhardtii could be carried out.

Part name

BBa_K3373012

BBa_K3373014

Type

Composite

Description

BSR_atzA-HA_atzB-HA_atzC-HA

BSR_NucA-TEVsite_UVR8-NtTEV_COP1-CtTEV

All the composite parts are linear DNA fragments that are the results of the digestion of level M plasmids by the type IIS BsaI restriction enzyme. It is these linear DNA fragments that are integrated into the genomic DNA of Chlamydomonas reinhardtii during the transformation.

BSR_atzA-HA_atzB-HA_atzC-HA ( BBa_K3373012 )

This part was designed to allow C. reinhardtii to degrade atrazine into cyanuric acid. It’s a multigenic construct containing three genes involved in the degradation pathway of the pesticide atrazine leading to cyanuric acid : atzA ( BBa_K3373001 ), atzB ( BBa_K3373002 ) and atzC ( BBa_K3373003 ). All these CDS are under the control of the PsaD promoter and terminator which allow a strong expression in C. reinhardtii. These three CDS are associated with an triple C-terminus HA tag allowing immunodetection of the proteins expressed. It also contains a Blasticidin resistance cassette (BSR) encoding for the Blasticidin S Deaminase in order to select the recombinant clones after transformation in C. reinhardtii.

BSR_NucA-TEVsite_UVR8-NtTEV_COP1-CtTEV ( BBa_K3373014 )

This part is a multigenic construct containing our three constructs for the operation of our kill-switch system adapted to C. reinhardtii. The CDS of the Nuclease A (NucA) fused to a double nuclear localization signal SV40 NLS ( BBa_K3373008 ) is associated at the N-terminus with the HAP2 transmembrane domain ( BBa_K3373007 ) and at the C-terminus with the recognition site for the TEV protease associated with a peptide linker ( BBa_K3373011 ). The CDS of UVR8 ( BBa_K3373005 ) is fused to the C-terminus domain of the TEV protease ( BBa_K3373010 ) with the GSAT peptide linker ( BBa_K3373006 ). The CDS of COP1 ( BBa_K3373004 ) is fused to the N-terminus domain of the TEV protease ( BBa_K3373009 ) with the GSAT peptide linker ( BBa_K3373006 ). All these CDS are under the control of the PsaD promoter and terminator which allow a strong expression in C. reinhardtii. It also contains a Blasticidin resistance cassette (BSR) encoding for the Blasticidin S Deaminase in order to select the recombinant clones after transformation in C. reinhardtii.