This study developed techniques for transforming the bacterium Halomonas Sp. 0-1 using electroporation in order to improve its synthesis of polyhydroxyalkanoates (PHAs), which are biodegradable plastics. Optimal conditions for Halomonas Sp. 0-1 growth and electroporation transformation were determined. Electroporation with 2 mm cuvettes, 1.5 kV voltage, and 1 hour recovery time achieved the highest transformation efficiency. This demonstrates Halomonas Sp. 0-1's potential for producing PHAs under salt-rich conditions, which could lower production costs and make PHAs more competitive with petroleum plastics.

1. Transformation of Halomonas Sp. 0-1 by Electroporation
For Improved Synthesis of Polyhydroxyalkanoates
Brian Grzeskowiak1, Joshua Harris1, Kouhei Mizuno2, Christopher Nomura1
1Department of Chemistry, State University of New York-College of Environmental Science & Forestry, 121 Jahn Laboratory, Syracuse, NY 13210 United States
2Department of Materials Science and Chemical Engineering, Kitakyushu National College of Technology, 5-20-1 Shii, Kouraminami-ku, Kitakyushu, Japan
Abstract
• Polyhydroxyalkanoates (PHAs) are biodegradable
polymers produced by a variety of bacterial species.
• Halomonas Sp. 0-1 is a newly discovered isolate capable
of producing PHAs in high-salt solutions. There is a
potential to reduce overall costs associated with PHA
production through this bacterium’s ability to synthesize
these polymers in conditions unfavorable to most other
microorganisms.
• Novel techniques were developed for the creation of
recombinant strains of Halomonas Sp. 0-1 using
electroporation.
• Optimal growth conditions occurred in LB media with
3.5% NaCl and pH 7.5 and kanamycin (50 µg/ ml) gave
best selection for screening.
• Optimal transformation occurred using 300 mM sucrose
as a wash and 2 mm gap cuvettes during electroporation
with a shock voltage of 1.5 kilovolts.
Introduction
• Petroleum-based plastics are created from a non-
renewable resource and don’t degrade quickly
(Figure 1).
• Interest in Polyhydroxyalkanoates (PHAs) has
grown over the years due to their potential as a
raw material for biodegradable plastics.
• A variety of bacteria species have evolved to
produce PHAs as a carbon reserve and electron
sink (Figure 2).
• PHA synthase genes have been isolated with the
potential to promote greater PHA yield through
transformation into different bacterial species.
• High production costs such as raw materials,
extraction techniques, and sterilization of growth
media, has prevented widespread use of PHA based
plastics.
Figure 1: Plastic accumulation at Kamilo Beach, Hawaii.
The Telegraph, 2009
Figure 2: (Keshavarz, T., & Roy, I., 2010)
• Halomonas Sp.0-1 is a new bacteria species
discovered off the coast of Japan.
• It is Gram-negative, an obligate aerobe, rod shaped
bacterium.
• It’s potential comes from it’s ability to produce
PHAs in high-salt solutions as it is a moderate
halophile (“salt loving”).
• It can produce the copolymer Poly(3-
hydroxybutyuric acid-co-3-hydroxyvaeric acid)
[(P(3HB-co-3HV)] which contains two different
monomers whose ratios can be altered to create
different mechanical properties similar to
traditional plastic (Figure 4).
Figure 4: P(3HB-co-3HV). Numbers of X and Y change mechanical
properties of the polymer.
Sigma-Aldrich
Figure 3: Various fatty acid carbon sources.
Methods
Freezer stock of
Halomonas Sp. 0-1 plated
(LB/3.5%NaCl/pH7.5)
Seed cultures
inoculated
(LB/3.5%NaCl/pH7.5)
2 mm cuvette with
culture and pBBR1-
MCS vectors used
(Figure 5).
Figure 5: pBBR-MCS vectors and their corresponding antibiotic resistance genes.
Wash
Electroporation performed (1.5 kV)
Recovered in liquid
media and plated on
antibiotic plates for
selection.
Results
Cuvette DNA Voltage Recovery time
Transformation efficiency
(CFU/µg DNA)
1 mm 75 ng 0.75 kV 1 hr 1.2 x 105
1 mm 75 ng 0.75 kV 2 hr 1.3 x 105
1 mm 75 ng 1.5 kV 1 hr 6.5 x 102
1 mm 75 ng 1.5 kV 2 hr 1.5 x 104
2 mm 75 ng 1.5 kV 1 hr 1.6 x 105
2 mm 75 ng 1.5 kV 2 hr 1.2 x 105
2 mm 75 ng 2.5 kV 1 hr 1.2 x 104
2 mm 75 ng 2.5 kV 2 hr 1.1 x 104
Table 1: Optimization of electroporation conditions
using Kan 50 for selection.
Table 2: Antibiotics tested
for selection of trans-
formants.
Electroporation theory
• Optimal media was found to contain LB/ 3.5% NaCl/ pH 7.5 (Similar to ocean conditions)
• Sucrose (300mM) washes had no effect on transformation efficiency.
• Electroporation variables that produced greatest transformation efficiency included 2
mm cuvettes, 1.5 kV charge deliveries, and 1 hr recovery in LB/ 3.5% NaCl/ pH7.5 liquid
media before plating (Table 1).
• Kanamycin produced the most definitive selection for transformation success (Table 2).
• Halomonas Sp. 0-1 showed natural resistance to Ampicillin.
• Restriction digests with Xba I was inconclusive, which could be
due to the effects of Halomonas on the restriction enzyme, such
as methylation (Figure 6).
• NanoDrop analysis showed presence of plasmid DNA with a
260nm/280nm abs of 2.07 (purity based on 260/280 absorbance >
1.0).
Conclusions
• Halomonas Sp. 0-1 was found to have the ability to
be transformed with plasmid DNA.
• PHA production in “salt-sterilized” cultures could
help reduce production costs on industrial levels.
• If the cost of PHA biodegradable plastics is lowered
enough it could economically compete with current
petroleum-based plastics.
• This was the first study to show successful
transformation in this species and the future would
involve polymer production and industrial sized
experiments.
300 mM
Sucrose
Antibiotic Conc. (µg/ ml)
Kan 50
Gm 20, 30
Cm 30, 50
Tc 10, 25
Amp 200
Figure 6: Agarose gel
electrophoresis of a restriction
digest on pBBR1-MCS containing
Hal. Sp.0-1.
References
• Mizuno K, Ilham M, Nakanomori S, Kihara T, Hokamura A,
Matsusaki H, and Tsuge T. Characterization of
polyhydroxyalkanoate synthases from Halomonas sp. O-1 and
Halomonas elongata DSM2581: Site-directed mutagenesis and
recombinant expression. Polymer Degradation and Stability 2014;
109: 416-423
• Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop II
RM, et al. Four new derivatives of the broad-host-range cloning
vector pBBR1MCS, carrying different antibiotic-resistance
cassettes. Gene 1995;166(1):175-6Acknowledgments
I owe everything to Joshua Harris for teaching me new molecular techniques in the lab , how to
use equipment new to me, showing me how a lab operates and how to work as a team. Thank
you Dr. Nomura for giving me the opportunity to work in a lab and Dr. Mizuno for isolating an
awesome new bacteria species allowing me to work on something never done before.