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Computational Molecular Biology
19
Interdisciplinary nature of the biopolymer science, are
created its challenges for large-scale studies in this
area that leads scientists towards affordable ways to
produce biodegradable polymers, Because of the high
cost of PHA production, would threaten the rapid
advance of the commercial exploitation of these
(Pandey et al., 2005). Finally, the PHAs have vast and
varied applications that Among these, medical
applications are the most viable as economically. Now,
with increasing interest and extensive research in this
field is ongoing, PHA's potential as the next
generation of environmentally friendly materials with
a wide range of applications are emerging. Due to the
small number of studies on genes involved in the
synthesis of PHA in the world and the potential of
phaC1
gene, in production of PHA and that gene is
new, transfer this gene into suitable expression vector
and transformation of that in
E. coli
bacteria in order
to expression of that, it is very important. Also, the
transfer of this gene with
phaD
and
phaG
genes to
bacteria and also transfer of this simultaneously to
several different bacterial species, can be a very good
approach to increase gene expression as more
efficiency and studies.
The PHA synthase
identification of other indigenous bacteria and effort
to produce this substance on large scale and
commercial is also very helpful.
3 Materials and Methods
3.1 Bacterial strains and cultivation conditions
The taxonomic characteristics of
Pseudomonas putida
KT2442 have been reported (Hume et al., 2009). Cells
were pre-cultured in 100-mL Erlenmeyer flasks
containing 25 mL Luria broth [(5 g/L yeast extract, 10
g/L tryptone, 5 g/L NaCl ) Merck Darmstadt,
Germany] for 24 h at 30
℃
in a rotary incubator at 150
rpm (Akhavan Sepahy et al., 2011). For production
and extraction of PHA, cells were cultivated in a
shaker incubator (180 rpm) at 30
℃
for 72 h in
500-mL flasks containing 100 mL of a
nitrogen-limited mineral salt (MS) medium. The
standard mineral salts medium is composed of (per
liter of distilled water): 1.269 g CH
3
CH
2
COOH, 600
mg MgSO
4
·7H
2
O, 160 mg NH
4
Cl, 100 mg
ethylenediamine tetraacetic acid (EDTA), 92 mg
K
2
HPO
4
, 45 mg KH
2
PO
4
, 70 mg CaCl
2
·2H
2
O and 2
mL of trace elements solution. The trace solution is
consisted of (per liter of distilled water): 1 500 mg
FeCl
3
·6H
2
O, 150 mg H
3
BO
3
, 150 mg CoCl
2
6H
2
O,
120 mg MnCl
2
·4H
2
O, 120 mg ZnSO
4
·7H
2
O, 60 mg
Na
2
MoO
4
·H
2
O, 30 mg CuSO
4
·5H
2
O and 30 mg of KI.
Filter-sterilized carbon sources were also added to the
medium (Watcharakul et al., 2012).
3.2 Sample preparation for FT-IR confirmation of
PHA
For qualitative and quantitative analysis of PHA, 7~10
mg of lyophilized cells or of isolated polyesters was
subjected to methanolysis in the presence of 15% (v/v)
sulfuric acid suspended in methanol. PHA was
extracted from lyophilized cells by chloroform in a
Soxhlet apparatus, and subsequently precipitated in 10
vol. methanol. The precipitate was dissolved in
acetone and was again precipitated in methanol in
order to obtain highly purified PHA. Purified PHA
was dissolved in chloroform and layered on the
KRS-5 window. After evaporation of the chloroform,
the PHA polymer film was subjected to FT-IR analysis
(Mai et al., 2004; Hong et al., 1999).
3.3 PCR Amplification Method
Total genomic DNA was isolated by standard
procedures using a Wizard Genomic DNA Purification
Kit (Promega, Madison, WI) (Sambrook et al., 1989).
PCR was performed in a Techne PCR Thermocycler
(Techne, England) and sequencing primers were
synthesized by PCR premix was purchased from
fermentas and was used according to the manufacturer’s
protocols. To amplify the PHA synthase gene,
phaC1
,
from genomic DNA of
Pseudomonas putida
, PCR was
performed with two specific primers, Forward primer:
ACAGATCAACAAGTTCTACATCTTCGAC, Reverse
primer GGTGTTGTCGTTGTTCCAGTAGAGGATGTC.
PCR conditions were as follows: 30 cycles of
denaturation at 94
℃
for 30 sec, annealing at 60
℃
for
30 second, elongation at 72
℃
for 1 min. The PCR
products were analyzed by agarose gel electrophoresis
in 1 × TBE (Tris-Borate-EDTA) buffer and gel was
stained with 0.5 ug/mL ethidium bromide.
3.4 Nucleotide sequence analysis
PCR-amplified
phaC1
gene with forward and reverse
primers was sequenced by the modified dideoxy chain
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