Part:BBa_K1720000

Human guanylate cyclase1,soluble, alpha 3 unit

This is a part used for coding soluble human guanylate cyclase subunit:Alpha 3.Alpha 3 unit interacts with a beta subunit to form the guanylate cyclase enzyme. Soluble guanylate cyclases(sGC) are heterodimeric proteins that catalyze the conversion of GTP to 3',5'-cyclic GMP and pyrophosphate. It is important for smooth muscle relaxation in the cardiovascular system. sGC contains an HNOX domain, which serves as a receptor for ligands such as nitric oxide, oxygen and nitrovasodilator drugs.There are several different isoforms of sGC subunit ,but the cardiovascular system abundant with alpha 3 unit and beta 3 unit.

We used lentiviral vector to transfected alpha 3 unit and beta 3 unit to HEK293 cells together with a mCherry reporter and vivo red fluorescence signal was observed under fluorescence microscope, it meant that this part was transfected into HEK293 cells successfully. Then we used qRT-PCR to observe transcriptional level of alpha 3 unit. Since soluble guanylate cyclases (sGC) are heterodimeric proteins that catalyze the conversion of GTP to 3',5'-cyclic GMP(cGMP) and pyrophosphate. The level of cGMP will be up regulated.e detected the cGMP concentration with Elisa Kit.The positive control was HEK293 cells that treat with Sodium Nitroprusside ,a NO donator that activate sGC and up regulate the level of cGMP. A negative control was made by transfecting an empty vector that does not contain sGC subunit. We used Elisa Kit to detect cGMP level. The results are as follow:

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 1498
  Illegal PstI site found at 1827
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 1498
  Illegal PstI site found at 1827
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 1498
  Illegal PstI site found at 1827
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 1498
  Illegal PstI site found at 1827
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1576
  Illegal SapI site found at 1446
  

Before we start the experiment, we designed an lentiviral vector that contains our part. and packaged the vector with lentivirus.

Vector Map：