Part:BBa_K1033001

4-coumarate ligase (4CL) with RBS

4-coumarate ligase (4CL) is an enzyme that catalyses the reaction from p-coumaric acid to 4-coumaroyl-coenzyme A (4-coumaryl-CoA). This enzyme is derived from the plant arabidhopsis thaliana, but exists in many other plants. [1]

In our project, we have been using it together with stilbene synthase, that produces resveratrol with the help of this enzyme.

Applications With the help of this enzyme one can produce 4-coumaryl-CoA which is an important precursor in many metabolic pathways. For example, it can be used to produce resveratrol with stilbense synthase. [2]

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1108
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1675
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1307
  

iGEM 2022 iBowu-China, new documentation (For Bronze)

Author: Enshi Xv

     In this year’s iGEM competition, SHSBNU_China team focused on anthocyanins production, especially delphinidin. 4CL gene is one of the key gene in the synthetic pathway of delphinidin, which catalyzed phenylalanine into 4-coumaryl coenzyme A.

We synthesized the sequence and constructed into plasmid pETDuet. </p> After transforming to E.coli BL21 and induced by IPTG overnight at 16 degree Celsius at various concentrations (0.25 to 2 uM), we lysed the bacteria and performed SDS-PAGE and we confirmed 4CL enzyme to be expressed successfully. Judged by the thickness of the SDS-PAGE band width and darkness, concentrations of iPTG doesn’t play a critical role in the expression of 4CL enzyme.

File:File:Bronze-1.jpg

Figure 1. The expression test for BBa_K1033001