Niels Jensen
Psilocybin and psilocin are the main alkaloids from many basidiomycetes in the genus Psilocybe. The molecular cloning of enzymes involved in their biosynthesis was attempted using an expression cloning strategy followed by a colony screening procedure for enzymatic activities. To establish a continuous supply of biomaterial, the influence of plant hormones, media, and culturing techniques on the growth and alkaloid content of Psilocybe tampanensis, Psilocybe cubensis, Psilocybe cyanescens, and Psilocybe azurescens was evaluated. An HPLC method for the simultaneous detection of psilocybin and psilocin was developed. Psilocybe tampanensis was found to produce sclerotia of a high alkaloid content on malt extract / yeast extract solid agar medium in good yield. A cDNA library was prepared from these sclerotia and inserted into the vector pYes2 using a methyl-cytosine / adapter ligation procedure. A cDNA synthesis method using an asymmetrically tailed vector-primer was not succesful. Amplification in E. coli yielded a total of 100,000 independent colonies in 20 plasmid pools. Screening procedures for all enzymes of the psilocybin biosynthesis have been developed. The yeast Saccharomyces cerevisiae was transformed and the resulting colonies were screened for tryptophan decarboxylase activity. The identification of positive clones was not possible due to the limited amount of time.
Submitted: June 1999
Sorry, but due to Fortunecity's file size and type restrictions the files had to be split and compressed.
Please download either the ZIPped PDF files:
Pages 1-35, 36-40, 41-46, 47-66
Or download the whole file DIPLOMA.RTF (11.9 MB) as a multiple-volume ARJ-compressed and ARJ-recompressed file. This file can be opened in MS-Word.
ARJ Compressed: 1, 2, 3, arj.exe (file compression program)
Download these files into one directory and extract the file DIPLOMA.RTF using the following commands in a DOS window:
ARJ x DA_ARJ.ARJ ARJ x DA_A01.ARJ ARJ x DA_A02.ARJ ARJ x -v DIPLOMA.ARJ
Please e-mail me under: Niels_H_Jensen@hotmail.com
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Last updated: August 29, 2003