pBIN61:α-AIC3

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The nucleotide sequence for the gene (aic3) encoding the α-AIC3 variant was obtained in silico via reverse translation and codon optimization of the α-AIC3 protein sequence [GenBank:AGB50990.1], as reported by Silva et al.	The aic3 gene was then excised from pUC18:α-AIC3 and cloned into the XbaI and BamHI sites of the pBIN61 binary expression vector, which was previously described by Bendahmane et al. [51] under the control of the constitutive CaMV 35S promoter and terminator to generate pBIN61:α-AIC3 that was used to transform A. tumefaciens strain C58C1 via electroporation. The cloning into the pBIN61 vector was confirmed by sequencing using the M13 forward primer and carried out by Beckman Coulter Genomics®. Cells were also transformed with empty vectors; these cells served as negative controls.

Upstream tips
The nucleotide sequence for the gene (aic3) encoding the α-AIC3 variant was obtained in silico via reverse translation and codon optimization of the α-AIC3 protein sequence [GenBank:AGB50990.1], as reported by Silva et al.
Protocol tips
The aic3 gene was then excised from pUC18:α-AIC3 and cloned into the XbaI and BamHI sites of the pBIN61 binary expression vector, which was previously described by Bendahmane et al. [51] under the control of the constitutive CaMV 35S promoter and terminator to generate pBIN61:α-AIC3 that was used to transform A. tumefaciens strain C58C1 via electroporation. The cloning into the pBIN61 vector was confirmed by sequencing using the M13 forward primer and carried out by Beckman Coulter Genomics®. Cells were also transformed with empty vectors; these cells served as negative controls.

Publication protocol

The nucleotide sequence for the gene (aic3) encoding the α-AIC3 variant was obtained in silico via reverse translation and codon optimization of the α-AIC3 protein sequence [GenBank:AGB50990.1], as reported by Silva et al. [26]. Codon optimization was performed with Gene Designer 2.0 software [50] based on the codon usage table for N. benthamiana species, available at Kazusa Codon Usage Database. The nucleotide sequence for the corresponding native signal peptide (MASSNLLSLALFLVLLTHANS) was also retrieved and codon-optimized. The final insert sequence was flanked by 5′-XbaI and 3′-BamHI restriction sites, and a Kozak consensus sequence (GCCACC) was inserted immediately upstream of the start codon. No restriction sites for XbaI and BamHI were detected within the CDS. SignalP 4.1 Server was used for signal peptide detection and validation. The sequence was synthesized de novo by Epoch Life Science® and cloned into the XbaI-BamHI cloning sites of the pUC18 vector to generate pUC18:α-AIC3. The aic3 gene was then excised from pUC18:α-AIC3 and cloned into the XbaI and BamHI sites of the pBIN61 binary expression vector, which was previously described by Bendahmane et al. [51] under the control of the constitutive CaMV 35S promoter and terminator to generate pBIN61:α-AIC3 that was used to transform A. tumefaciens strain C58C1 via electroporation. The cloning into the pBIN61 vector was confirmed by sequencing using the M13 forward primer and carried out by Beckman Coulter Genomics®. Cells were also transformed with empty vectors; these cells served as negative controls.

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Download the product protocol from Séverine Lacombe, IRD, CIRAD, Université Montpellier, Interact for pBIN61:α-AIC3 below.

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