Team:IISER-Bhopal-India/Results
Gene Splicing by Overlap Extension PCR
Parts and vector confirmation:
BioBrick parts obtained from iGEM Repository and vectors ordered from AddGene were confirmed by their transformation into competent DH5α strain of E.coli and subsequent plasmid isolation from the obtained colonies.
(b) Isolation and purification of pGKB plasmid - A Pichia pastoris expression vector pGKB (3506bp) procured from AddGene was isolated and purified by miniprep and run on 1% agarose gel for validation
PCR Amplification of parts to be cloned:
PCR amplification of sMMO D and sMMO X genes using appropriate primers (IDT) with overhangs containing restriction sites necessary for cloning into pGKB along with kozak sequence for protein expression in Pichia was done.
Gel Electrophoresis was done for the amplified parts and DNA bands were visualized. Bands of proper sizes were observed which confirmed the size of the parts.
sMMO D and sMMO X cloned in pGKB Vector:
Genes for MMO subunits-sMMO D and sMMO X- obtained after PCR Amplification and pGKB Vector were digested with Restriction Enzymes NgoMIV and SalI at 37ºC overnight. Digested DNA was purified by Gel Elution purification and ligation was setup followed by transformation into competent DH5α E.coli cells. Plasmid was isolated from the obtained colonies and confirmed by Agarose Gel Electrophoresis.
The cloned plasmid sMMO D_pGKB (pGKB with sMMO D gene flanked by GAP Promoter and AOX Terminator) was confirmed by double digestion with NgoMIV and SalI and sMMO X_pGKB (pGKB with sMMO X gene flanked by GAP Promoter and AOX Terminator) with SacII and EcoRI. Bands of appropriate sizes were observed after Agarose Gel Electrophoresis which confirmed the cloning of sMMO D and sMMO X in pGKB.
PCR Amplification of DNA fragments for SOEing PCR:
Genes for sMMO D and GAP Promoter were PCR amplified from sMMO D_pGKB recombinant plasmid with primers necessary for introducing the overlap region required for SOEing PCR. This confirmed the cloning of sMMO D in pGKB as well. Also, genes for sMMO X and AOX Terminator were PCR amplified from sMMO X_pGKB recombinant plasmid similarly. These were confirmed by observing bands of appropriate sizes after Agarose Gel Electrophoresis.
SOEing PCR for sMMO D and AOX Terminator:
sMMO D and AOX Terminator were joint by homologous recombination using SOEing PCR. The amplified product was visualised and verified by gel electrophoresis.
SOEing PCR for sMMO X and GAP Promoter:
sMMO X and GAP Promoter were joint by homologous recombination using SOEing PCR. The amplified product was visualised and verified by gel electrophoresis.
Cloning of sMMO D+AOX Terminator in pGKB Vector:
sMMO D+ AOX Terminator construct obtained after PCR Amplification and pGKB Vector were digested with restriction enzymes NgoMIV and KpnI at 37ºC overnight. Digested DNA was purified by Gel Elution purification and ligation was setup followed by transformation into competent DH5α E.coli cells. The plasmid was isolated from the obtained colonies and run on a gel by agarose gel electrophoresis. The cloned plasmid sMMO D+AOX Terminator_pGKB was visualized and verified by observing a band of expected size by gel electrophoresis. The cloned plasmid was further confirmed by double digestion of the plasmid with NgoMIV and KpnI after which bands of appropriate sizes were observed.
Cloning of sMMO X+GAPP in sMMO D+AOXT_pGKB Vector:
sMMO X+GAPP construct obtained after PCR Amplification and sMMO D+AOXT_pGKB Vector were digested with restriction enzymes KpnI and SalI at 37’C overnight. Digested DNA was purified by Gel Elution purification and ligation was setup followed by transformation into competent DH5α E.coli cells. Plasmid was isolated from the obtained colonies and run on gel by agarose gel electrophoresis.
The cloned plasmid sMMO D+AOXT+sMMO X+GAPP_pGKB was visualized and verified by observing band of expected size by gel electrophoresis.
The cloned plasmid was further confirmed by double digestion of the plasmid with KpnI and SalI after which bands of appropriate sizes were observed.
Antibiotic Sensitivity Assay
Antibiotic Sensitivity Assay for Pichia pastoris for G418:
Pichia pastoris was grown in different concentrations of the antibiotic G418 to determine the optimum concentration of the antibiotic to be used for selection of the transformed yeast. O.D.600 of the culture was recorded at different time points with antibiotic concentrations ranging from 100μg/ml to 500μg/ml and plotted against time. The results obtained were analysed and 300μg/ml was found to be optimal for selection of the transformed yeast as the growth of the original untransformed strain was restricted in that concentration. Further yeast transformations were done using plates with 300μg/ml G418. 
Alternate cloning strategy
Cloning of P2A Insert 1 in pGKB vector:
Genes for MMO subunits - sMMO B, sMMO Y and sMMO D- synthesized as gBlocks from IDT with 6xHis tags tailing each subunit flanked by the Kozak sequence in the front and intermediate P2A sequences (Insert Name: P2A1) and pGKB Vector were digested with Restriction Enzymes EcoRI and KpnI at 37ºC overnight. Digested DNA was purified by Gel Elution purification and ligation was setup followed by transformation into competent DH5α E.coli cells. Plasmid was isolated from the obtained colonies and confirmed by Agarose Gel Electrophoresis. The cloned plasmid P2A1_pGKB (pGKB with P2A1 insert flanked by GAP Promoter and AOX Terminator) was confirmed by double digestion with EcoRI and KpnI. Bands of appropriate sizes were observed after Agarose Gel Electrophoresis which confirmed the cloning of P2A1 Insert in pGKB.
Part Submission:
Cloning of P2A1 and P2A2 in pSB1C3
P2A1(sMMO C, sMMO D and sMMO Y) and P2A2(sMMO B, sMMO X and sMMO Z) parts were submitted, cloned into pSB1C3, for the silver medal criteria.
