Difference between revisions of "Part:BBa K4117222:Experience"

Revision as of 19:32, 11 October 2022

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Applications of BBa_K4117222

Experiment: 1.1synthesis of pathways to metabolism THC We design the Plasmids and had the company GENEWIZ synthesized them. 1.2 Construction of pathway to verify UGT1A3 extracellular secretion We obtain the gene fragments of UGT1A3, ompT and His-tag by PCR from the UGT1A3. After the completion of PCR, electrophoresis is performed to determine the size of DNA, and then DNA fragments are recovered. Finally we insert it into the pET-Duet1 downstream of the T7 promoter and upstream of the T7 terminator to verify the external secretion of UGT1A3 under IPTG induction. 1.3Verification of imported gene pathways We transform E. coli BL21(DE3) competent cells with the prepared plasmid for overnight culture and preserve the engineered bacteria in glycerol and stored them at -80℃ for standby. 1.4 Feasibility experiments on IPTG induction We select E. coli BL21(DE3) IPTG-induction E. coli BL21(DE3) strain and add 100μL 500mmol/L IPTG for each 50mL bacterium to induce the expression of UGT1A3 overnight. The fusion expression proteins are purified by his-tag protein extraction kit and verified by SDS-PAGE gel. Result: We construct the THC metabolism pathway of UGT1A3 which induced by THC. 1. The verification of plasmids for correctness. We insert the gene fragments of UGT1A3, ompT and His-tag into the pET-Duet1 downstream of the t7 promoter and upstream of the T7 terminator, and verify the bacterial colony PCR products by gel electrophoresis to initially determine the correctness of the plasmid construction. After that we extract the plasmids and verify the correct construction of the plasmids by sequencing them by the company. 2. Protein expression, purification verification. The fusion expression proteins are purified by His-Tag protein extraction kit and verified by SDS-PAGE gel. The results show that the proteins are successfully expressed.

User Reviews

UNIQ12ef324a358a5257-partinfo-00000000-QINU UNIQ12ef324a358a5257-partinfo-00000001-QINU

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 ===Applications of BBa_K4117222===
-UGT1A3 plays an significant role in metabolic section, working upon the sensing section. Tetrahydrocannabinol (Δ9 -THC), the primary psychoactive ingredient in marijuana, is subject to cytochrome P450 oxidation and subsequent UDP-glucuronosyltransferase(UGT)-dependent glucuronidation. Many studies have shown that CYP2C9 and CYP2C19 are the primary enzymes responsible for these cytochrome P450-dependent oxidations.The specific human UGTs are responsible for classic cannabinoid metabolism, and among them, the highest activity toward THC-COOH was observed with UGT1A3.
+Experiment: 1.1synthesis of pathways to metabolism THC We design the Plasmids and had the company GENEWIZ synthesized them. 1.2 Construction of pathway to verify UGT1A3 extracellular secretion We obtain the gene fragments of UGT1A3, ompT and His-tag by PCR from the UGT1A3. After the completion of PCR, electrophoresis is performed to determine the size of DNA, and then DNA fragments are recovered. Finally we insert it into the pET-Duet1 downstream of the T7 promoter and upstream of the T7 terminator to verify the external secretion of UGT1A3 under IPTG induction. 1.3Verification of imported gene pathways We transform E. coli BL21(DE3) competent cells with the prepared plasmid for overnight culture and preserve the engineered bacteria in glycerol and stored them at -80℃ for standby. 1.4 Feasibility experiments on IPTG induction We select E. coli BL21(DE3) IPTG-induction E. coli BL21(DE3) strain and add 100μL 500mmol/L IPTG for each 50mL bacterium to induce the expression of UGT1A3 overnight. The fusion expression proteins are purified by his-tag protein extraction kit and verified by SDS-PAGE gel.
+Result: We construct the THC metabolism pathway of UGT1A3 which induced by THC. 1. The verification of plasmids for correctness. We insert the gene fragments of UGT1A3, ompT and His-tag into the pET-Duet1 downstream of the t7 promoter and upstream of the T7 terminator, and verify the bacterial colony PCR products by gel electrophoresis to initially determine the correctness of the plasmid construction. After that we extract the plasmids and verify the correct construction of the plasmids by sequencing them by the company. 2. Protein expression, purification verification.
+The fusion expression proteins are purified by His-Tag protein extraction kit and verified by SDS-PAGE gel.
+The results show that the proteins are successfully expressed.
 ===User Reviews===