Team:Toulouse INSA-UPS/Parts

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Parts

To construct our parts, we used different approaches: some were synthesized by IDT or Twist bioscience (which allowed us to obtain codon-optimized sequences for Saccharomyces cerevisiae and Synechococcus elongatus), others were obtained by PCR amplification from the genomes of Synechococcus elongatus or from plasmids. The parts were then built in the desired vector by In-Fusion assembly.

Here is a summary table of all the parts iGEM Toulouse INSA-UPS 2021 designed this year. Key information is added such as length, the current status (works, failed, successful cloning or in progress), and RFC compliance (parts with "*" cannot be associated with a RFC according to restriction site rules. Nonetheless, each individual element constituting these parts can be associated with at least one RFC).

If you want to know more about our parts or any inquiry, feel free to contact us at igem.toulouse@gmail.com

Type	Purpose	Accession number	Diagram	Significance in the project	Length (bp)	Status	RFC
Basic	Linalool and dihydro-β-ionone induction system and expression in S. cerevisiae	BBa_K3930000		+++	8777	Works	*
Basic	β-ionone induction systems and expression in S. cerevisiae (enzymatic fusion)	BBa_K3930001		+++	7748	Works	*
Basic	β-ionone induction systems and expression in S. cerevisiae	BBa_K3930002		+++	8341	Works	*
Basic	α-ionone induction systems and expression in S. cerevisiae (enzymatic fusion)	BBa_K3930003		+++	6260	Works	*
Basic	Up Integrative sequence to target locus XII-1 of S. cerevisiae genome	BBa_K3930004		+	516	Works	1000
Basic	Down integrative sequence to target locus XII-1 of S. cerevisiae genome	BBa_K3930005		+	347	Works	21
Basic	Inducer Z3EV of the estradiol promoter	BBa_K3930006		++	2021	Successful cloning	21
Basic	Estradiol Inducible promoter	BBa_K3930007		++	625	Successful cloning	10, 12, 21, 23, 1000
Basic	Gene coding for the Linalool synthase	BBa_K3930008		+++	1689	Successful cloning	21, 1000
Basic	Gene coding for the DBR1 enzyme	BBa_K3930009		+++	1044	Works	12, 21, 1000
Basic	Hygromycin selective cassette	BBa_K3930011		+	1837	Works	21
Basic	Up integrative sequence to target locus X-3 of S. cerevisiae genome	BBa_K3930012		+	437	Works	10, 12, 21, 23, 25, 1000
Basic	Down integrative sequence to target locus X-3 of S. cerevisiae genome	BBa_K3930013		+	521	Works	1000
Basic	Doxycycline inducible promoter	BBa_K3930014		++	1008	Issues	10, 12, 23, 25, 1000
Basic	Constitutive promoter TEF1	BBa_K3930015		++	398	Works	10, 12, 21, 23, 25
Basic	Fusion between CrtY and phCCD1 with a LGS linker to produce β-ionone	BBa_K3930016		+++	2847	Works	10, 12, 23, 25, 1000
Basic	Fusion between phCCD1 and the fyn anchor with linker to produce β-ionone	BBa_K3930017		+++	1722	Works	10, 23, 25, 1000
Basic	Gene coding for the lycopene cyclase CrtY	BBa_K3930018		+++	1146	Works	10, 12, 21, 23, 25, 1000
Basic	Inducer rtTA for the TetO7 promoter	BBa_K3930019		++	1446	Successful cloning	10, 12, 21, 23, 25, 1000
Basic	G418 selective cassette for yeast genome integration event	BBa_K3930020		+	1263	Works	10, 12, 21, 23
Basic	Up integrative sequence to target locus XII-4 of S. cerevisiae genome	BBa_K3930021		+	501	Works	10, 12, 23, 25 1000
Basic	Down integrative sequence to target locus XII-4 of S. cerevisiae genome	BBa_K3930022		+	435	Works	10, 12, 23, 25, 1000
Basic	Galactose inducible promoter	BBa_K3930023		++	442	Works	10, 12, 21, 23, 1000
Basic	Fusion between LCYe and ofCCD1m with a LGS linker to produce α-ionone	BBa_K3930024		+++	3486	Works	10, 12, 23, 25, 1000
Basic	Nourseothricin selective cassette	BBa_K3930025		+	1196	Works	10, 12, 21, 23
Basic	3,6-nonadienal induction system and expression in S. elongatus	BBa_K3930026		+++	8815	Successful cloning	*
Basic	Integrative right site in the NSI locus of the S. elongatus genome	BBa_K3930027		+	780	Works	21
Basic	Integrative left site in the NSI locus of the S. elongatus genome	BBa_K3930028		+	871	Works	21, 1000
Basic	IPTG and theophylline inducible promoter	BBa_K3930029		++	155	Successful cloning	10, 12, 23, 25, 1000
Basic	Gene coding for the 9-lipoxygenase	BBa_K3930030		+++	2589	Successful cloning	10, 12, 23, 25
Basic	Gene coding for the 9-Hydroperoxide lyase	BBa_K3930031		+++	1446	Successful cloning	10, 12, 23, 25
Basic	Spectinomycine selective cassette	BBa_K3930032		+	1339	Works	10, 12, 21, 23

Former Part characterizations

The project developed this year by the iGEM team of Toulouse has created many parts for metabolic engineering. Others parts we used were already present in the iGEM registry. Here are some we used and for whose we gathered new information:

BBa_K1969005: this part corresponds to the pCUP1 promoter, inducible by the addition of copper in the medium. This part had not been characterized before, but the team showed this year that it is functional for expression in S. cerevisiae.

BBa_K2117000 and BBa_K2117005: those parts correspond to the pTEF1 promoter. They had been characterized before for expression in Y. lipolitica, but the iGEM Toulouse 2021 team showed that it is also functional for expression in S. cerevisiae.

BBa_K3629011: this part corresponds to the nsrR resistance gene, useful for transformants selection. This part had not been characterized before and its expression was supposed to be optimized for the chassis Y. lipolitica. We demonstrated here that this part, once codon optimized for S. cerevisiae, is also functional in this chassis.

BBa_K1323012: this part corresponds to the spcR resistance gene and had not been characterized before. We showed this year that it is functional for expression in the cyanobacterium S. elongatus.

BBa_K1313004: this part corresponds to the neoR resistance gene, useful for E. coli transformants selection with neomycin. We demonstrated here that this part is also functional in S. cerevisiae for selection on G418.

BBa_K1969004: this part corresponds to the Gal1 promoter, lacking the kozak sequence. No experience results were obtained for it. We demonstrated here this version of the Gal1 promoter is functional in S. cerevisiae.

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