Transformation of E.coli with Hydrogenase from Pyrococcus furiosus
Shiran Zhavian & Daniel Turman
Abstract
Materials and Methods
Results
The utilization of the planet’s most ubiquitous organic material may provide the
only solution to declining energy resources and the efficient conversion of
cellulose and glucose for hydrogen production can provide the way. The purpose of
our experiment is to explore the possibility of transforming ordinary bacteria with
the hydrogenase enzyme found in the hyperthermophile Pyrococcus furiosus
which not only resides in one of the most hostile environments on Earth but also
produces hydrogen as a by product. Our methods in approaching this problem
included PCR amplification, restriction endonuclease digestion, DNA purification,
ligation and transformation. We were successful in isolating the four sub units of
the hydrogenase enzyme from Pyrococcus furiosus. Future studies will determine
if the subunits expressed in E. coli can be used for Hydrogen production.
The pET-22b(+) vector (Cat. No. 69744-3) carries an N-terminal pelB signal sequence for potential
periplasmic localization, plus optional C-terminal His•Tag® sequence. Unique sites are shown on the
circle map. Note that the sequence is numbered by the pBR322 convention, so the T7 expression
region is reversed on the circular map. The cloning/expression region of the coding strand transcribed
by T7 RNA polymerase is shown below. The f1 origin is oriented so that infection with helper phage
will produce virions containing single-stranded DNA that corresponds to the coding strand. Therefore,
single-stranded sequencing should be performed using the T7 terminator primer (Cat. No. 69337-3).
SUBUNITS
pET-22b
A22 B22 C22 D22
A B C
D
DNA PURIFICATION
A B C D
pET-22b vector
Introduction
Pyrococcus furiosus ("the rushing fireball") is found in the marine sand
surrounding sulfurous volcanoes. It is anaerobic (lives without oxygen), and is
capable of growing at temperatures ranging from 70-103 C and at a pH ranging from
5-9. P. furiosus is remarkable because it is able to maintain chromosomal integrity
at temperatures up to 103 C with very little accumulation of DNA breaks. It also is
highly resistant to radiation and can withstand doses up to 1.5 kGy. These
organisms also possess an array of highly thermostable enzymes that could prove
to be important biocatalysts.[1]
DNA PURIFICATION
DNA PURIFICATION
pET-22b
LIGATION
Potential ligation
of subunit A.
A D
PCR
Pyrococcus furiosus
RESTRICTION ENDONUCLEASE
DIGESTION
Hydrogenase
Many microorganisms contain an enzyme or system of enzymes, called
"hydrogenase," which activates molecular hydrogen. Most methods for the assay
of hydrogenase activity measure the rate of reduction of some compound by
molecular hydrogen.[2]
Hydrogenases are enzymes that catalyze the equilibrium reaction
H 2 ↔ 2H+ + 2e-.[3]
There are three different types of hydrogenases classified based on the chemistry
of their active sites. They are: 1) Nickel-Iron ([NiFe]), 2) Iron only ([FeFe]), and 3)
Metal free, which actually contains one iron ([Fe]). Typically, organisms that
catalyze the oxidation of H2 contain [NiFe]-hydrogenases, and organisms that
reduce H+ to H2 contain [FeFe]-hydrogenases. Not much is known about the [Fe]hydrogenase (as it is the topic of this report). Hydrogenases play a role in several
microbial metabolic pathways (mentioned later) [4]. Hydrogenases are primarily
found in prokaryotic microorganisms, but also occur in some eukaryotes [5].
The energy yielded from oxidation of molecular hydrogen, when compared with
oxidation (combustion) of similar weights of known high energy compounds (i.e.
gasoline, coal, natural gas), is the highest. In fact, 1 kilogram of H2 has 2.8 times as
much energy as a kilogram of gasoline and 2.1 times as much energy as 1 kilogram
of natural gas [6]. The energy obtained from the combustion of H2 is 286 kJ/mole of
H2 (an extremely high value).
Subunit A colony
Subunit D colony
pET-22b colony
Conclusion
DNA PURIFICATION
LIGATION
We were successful in amplifying the genes for the four subunits which constitute the
building blocks of the hydrogenase enzyme from Pyrococcus furiosus and our LB+Amp
plates yielded colonies following transformation of E.coli with these four different subunits.
However, ligation was not completely successful. The method for ligation was altered to
achieve the linking. We are in the process of final transformation. Future studies will
determine if the subunits expressed in E. coli can be used for Hydrogen production.
References
Hydrogen combustion yields only energy and water and uses no carbon, so it can't
emit carbon-containing gasses or other harmful pollutants. In fact, hydrogen is the
cleanest of currently known fuel sources. Another attractive feature of H2 as a fuel
is that it is renewable. Furthermore, hydrogen is the most abundant element in the
earth and its atmosphere [7].
TRANSFORMATION
[1]
http://www.ebi.ac.uk/2can/genomes/archaea/Pyroco
ccus_furiosus.html
[2] http://www.pnas.org/content/42/4/180.full.pdf
[3]Adams and Steifel, 1998
[4] Zannoni, 2005
[5] Adams and Steifel, 1998
[6] Kushnir, 2000
[7]
http://dolly.biochem.arizona.edu/Bioc462b_Honors_
Spring_2009/mkarolak/index.html)
Acknowledgements
David Moloney, PhD
Kristen La Magna, MAT
Jennie Williams, PhD
Delon Callender, BA
Stancy Joseph, PhD
David Bynum, PhD
Judy Nimmo
Debbie Pelio
This research was supported by
NIH Bridges to the Baccalaureate
grant #GM50070.