Difference between revisions of "Part:BBa K4117888:Experience"

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===Applications of BBa_K4117888===
 
===Applications of BBa_K4117888===
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“T7 promoter+ PmrA+ RBS+ PmrB+ rnaB T1 terminator+ pmrC promoter” is the THC sensing pathway. To alert vaccinators that they have ingested THC, we design a THC sensing system. In this pathway, anti-THC can specifically bind to THC, and then promote PmrB phosphorylation of PmrA. PmrC promoters activate phosphorylated PmrA proteins and initiate transcription to express pigment proteins. Pigment proteins can be excreted in the stool, acting as a warning.
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==== Experiment: ====
  
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Outer membrane protease T (OmpT) is a 33.5 kDa endoprotease located on the outer membrane of Escherichia coli. OmpT signal peptide is an excretion tag linked on the N-terminus of protein. It can transport proteins across the outer membrane with the help of OmpT.  
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1.Synthesis of pathways to metabolism Δ9-THC.   We design the plasmid pUGT1A3 which contains the DNA sequence of UGT1A3 and had the company GENEWIZ synthesized them.
  
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UGT1A3 in this pathway 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. In Mazur’s study, the specific human UGTs are responsible for classic cannabinoid metabolism, and among them, the highest activity toward THC-COOH was observed with UGT1A3.
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2.Construction of pathway to verify UGT1A3 extracellular secretion.    We obtain the gene fragments of UGT1A3, ompT and His-tag by PCR from the pUGT1A3. 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.
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3.Verification 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.  
  
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Thus, in our system, Δ-9-ThC is hydroxylated by CYP2C9(BBa_K4117002) and CYP2C19(BBa_K4117022), remaining psychoactive. The major product of CYP2C9 and CYP2C19 metabolism, 11-OH-Δ-9-ThC, was further oxidized to THC-COOH. Then UGT1A3, which catalyzes the glucuronidation of the former substrate, increases the metabolite's water solubility, thereby facilitating excretion into the urine.
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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.
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==== Result: ====
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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.
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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 protein can be successfully expressed.
  
 
===User Reviews===
 
===User Reviews===

Latest revision as of 08:37, 12 October 2022


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Applications of BBa_K4117888

Experiment:

1.Synthesis of pathways to metabolism Δ9-THC. We design the plasmid pUGT1A3 which contains the DNA sequence of UGT1A3 and had the company GENEWIZ synthesized them.

2.Construction of pathway to verify UGT1A3 extracellular secretion. We obtain the gene fragments of UGT1A3, ompT and His-tag by PCR from the pUGT1A3. 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.

3.Verification 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.

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:

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 protein can be successfully expressed.

User Reviews

UNIQ70638ef076c69ccb-partinfo-00000000-QINU UNIQ70638ef076c69ccb-partinfo-00000001-QINU