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Identification and Determination of nucleotides involved in the process of
Biosynthesis of Radicicol from Pochonia chlamydosporia
Jia Zeng, Arumon Mukherjee
Biological and Irrigation Engineering Department, Utah State University
Contact: [email protected]
Introduction
Fungal polyketides sharing the resorcylic acid lactone (RAL) scaffold
display an impressive array of biological activities. Radicicol, one of
the fungal polyketides, is produced by Pochonia chlamydosporia. It
inhibits the Hsp90 molecular chaperone, another important target for
cancer chemotherapy.
Recently, gene clusters for biosynthesis of
radicicol from Pochonia chlamydosporia
were sequenced. However, the function
of each enzyme is still waiting for be
analyzed, which would interprets the
mechanism of radicicol synthesis process.
One of the significant differences between fungal and bacteria genes is
that the introns exist in the genomic DNA of fungi. When
heterologously expressed in E.coli cells, we must remove the introns
which can not be recognized by prokaryote. Therefore, Synthesize the
cDNA from mRNA is an effective method. In order to identify the
concentration and purity of mRNA and cDNA, NanoDrop is involved
in this experiment.
Experimental Procedure
7kb
Strains and culture condition: Pochonia chlamydosporia was obtained from
Agricultural Research Service Culture Collection of USDA and was
maintained on Difco potato dextrose broth (PDB) media. Incubated it at 28℃
for 4 days.
mRNA extraction: mRNA was isolated from P. chlamydosporia grown for 4
days in PDB media using a RNeasy Plant Mini Kit (Qiagen).
1. Extract the mRNA and reverse-trasnlate to cDNA from P. chlamydosporia.
2. Determine the concentration and purity of mRNA and DNA samples..
3. Clone the functtional genes by PCR.
cDNA cloning: SuperScript. III FirstStrand Synthesis System for RT-PCR Kit
(invitrogen) was used for reverse
transcription and synthesize the cDNA
from mRNA.
sample 1: the mRNA extracted from P. chlamydosporia.
sample 2: Purified mRNA by adding DNAase to remeove
the remaining genomic DNA
sample 3: cDNA Reverse-Translated from mRNA.
sample 4: PCR product of rdc1 genes, Using cDNA as template
sample 5: Bovine Serum Albumin (BSA) as protein comparison group
TM
NanoDrop
Sample measurement = 0.5 – 2.0 ul
We managed to clone those related functional genes from
cDNA of P.chlamydosporia which were reverse-translated
from mRNA. Using NanoDrop, we tested the purity and
determined the concentration of the extracted mRNA and
reverse-translated cDNA, by comparing the results of UV
absorption. The mRNA and cDNA can be used for the further
research such as PCR and protein expression in the future.
Future Work
2. Research on the property of the kinetic chacractors
High accuracy and high reproducibility
Results and Discussion
.
Patented sample retention technology between two optical fibres
Sample ID
Concentration
A260
A280
260/280
Principle:
Sample 1
154.47 ng/ul
3.089
1.489
2.07
A= ε * b * c
Sample 2
104.55 ng/ul
2.091
1.055
1.98
A = absorbance (A)
Sample 3
866.89 ng/ul
17.338
10.611
1.63
ε = Molar absorptivity coefficient (liter/mol-cm)
Sample 4
24.87 ng/ul
0.497
0.288
1.73
Sample 5
4.92 mg/ml
-
-
0.68
c = Analyte concentration (moles/liter or molarity)
Radicicol
1. Express rdc genes in E.coli cells.
NanoDropTM 1000
b = Pathlength (cm)
The compounds in P.
chlamydosporia check
by HPLC
supernatant
Conclusions
Identify all the above 5 samples and determine their concentration by NanoDrop.
Beer-Lambert equation
The PCR result of rdc1
mycelia
Identification and determination by NanoDrop:
Objectives
Results and Discussion
Table represents the different concentrations and
purity of the samples 1 – 5 using NanoDrop. Every
sample was tested twice and the mean value was
taken. It is clear that Sample 1 consisted of a
mixture of mRNA and g-DNA. After further
purification by adding DNAase, the result showed
that Sample 2 consisted of pure mRNA giving a
260/280 ratio of 1.98. From Sample 2, we reversetranslate it to cDNA, giving a 260/280 ratio of 1.63.
The cDNA from Sample 3 was used as a template
for PCR cloning. This gene is our Sample 4, which
is a pure DNA. Lastly, after recalibrating the
NanoDrop, we measured the purity of Bovine
Serum Albumin.
of rdc genes in vitro.
3. Synthesize different active compounds by different
combination of enzymes.
Acknowledgement
1. We are very thankful to Dr. Jixun Zhan for
providing the strain and extraction kit.
2. We are also very thankful to Dr. Charlie Miller
for providing us the NanoDropTM.