Coding

Part:BBa_K1181000

Designed by: Sebastian Roy, Maja Rennig   Group: iGEM13_Frankfurt   (2013-09-25)

Kaurenoic Acid Hydroxylase

This gene codes for an enzyme that catalyzes the reaction from ent-Kaurenoic acid to steviol. This is a precursor of the sweetener stevia. To perform this step NADPH is required.

Some data:

  • Reaction: ent-kaurenoic acid + NADPH + H+ + O2 -> steviol + NADP+ + H20
  • Km value: 11.1
  • pH optimum: 7.5 - 7.8
  • Maxima of absorption spectrum: 337 and 448 nm

Keun Ki Kim, Yoshihiro Sawa, Hitoshi Shibata, Hydroxylation ofent-Kaurenoic Acid to Steviol inStevia rebaudianaBertoni—Purification and Partial Characterization of the Enzyme, Archives of Biochemistry and Biophysics, Volume 332, Issue 2, 15 August 1996, Pages 223-230, ISSN 0003-9861, http://dx.doi.org/10.1006/abbi.1996.0336. (http://www.sciencedirect.com/science/article/pii/S0003986196903367) Keywords: ent-kaurenoic acid; hydroxylase; stevia sweetener; steviol; purification

Because of financial reasons we decided to benefit from a special offer (gBlocks) and ordered our gene encoding the ent-kaurenoic acid hydroxylase (KAH) in four parts that should be assembled via Gibson Assembly. The first three parts contain 500 bp (KAH1/ KAH2/ KAH3) and the fourth one has a size of 260 bp (KAH4). First of all, we ran PCRs with corresponding primers for Gibson Assembly. The result is shown below.

<img href="http://img12.imageshack.us/img12/3126/n3uu.jpg" alt="PCR of KAH gBlock Fragments" />

Afterwards, we performed Gibson Assembly with our four constructs. As it can be seen in the picture below, the assembly of KAH1 and KAH2 (product of 1000 bp) and the assembly of KAH3 and KAH4 (product of 760 bp) was successful.

<img href="http://img837.imageshack.us/img837/2796/nigq.jpg" alt="Gibson Assembly of KAH1 and KAH2 and Gibson Assembly of KAH3 and KAH4" />

But because of unknown reason the assembly of our new constructs KAH1/2 and KAH3/4 did not work out. For this reason, we were forced to synthesize our KAH gene as a whole.

The synthesized gene was transformed into E. coli DH5alpha and the plasmid containing our gene of interest was prepared. The gene was amplified via PCR with primers for gap repair cloning based on the principle of homologue recombination in yeast. As it is seen in the picture below the KAH gene was amplified successfully (1760 bp). With gap repair cloning we assembled our seconds plasmid, called the steviol plasmid, in yeast. As far as we can say, the assembly was successful as a lot of yeast colonies were growing. Due to a lack of time, no further characterisation and verification was performed with the assembled plasmid. <img href="http://img713.imageshack.us/img713/575/afx3.jpg" alt="PCR fragments for gap repair in yeast" />


For the construction of the KAH BioBrick we digested the KAH gene and the pSB1C3 backbone with EcoRI and PstI, performed ligation and transformation into E.coli DH5alpha. In the picture below the result of the transformation is shown. The preperation of the assembled plasmid accidentially failed and the construction of the BioBrick could not be proofed successfully in time. <img href="http://img51.imageshack.us/img51/7892/8jj1.jpg" alt="BioBrick Plates" />


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 293
    Illegal BglII site found at 1095
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4
    Illegal BsaI site found at 798


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