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Inactivation of the CpcB light-harvesting protein in Synechococcus sp. PCC
7002 cyanobacteria for increased cell density and bioproduct yields
McNair Scholar: Franki Mayer
Faculty Mentor: Dr. Toivo Kallas
Department of Biology University of Wisconsin Oshkosh
E. coli transformant colonies carrying a
plasmid for cpcB gene inactivation
Constructed ∆cpcB Inactivation Plasmid
Abstract
A growing concern worldwide is the continued use of products and fuels derived from
petroleum. These resources are limited, which presents the problem of finding
suitable replacements that need to be sustainable, renewable, and cost effective. One
such alternative to petroleum products that is currently being explored is isoprene
(C5H8), a precursor for numerous terpene products including synthetic rubber,
pharmaceuticals, plastics, and biofuels. One approach to the production of bioproducts is to use cyanobacteria, which are microalgae that derive energy from
sunlight and carbon from CO2.
Our group had genetically modified the
cyanobacterium Synechococcus sp. PCC 7002 to produce isoprene. However, for this
‘photo-isoprene’ to become a marketable option for replacing petroleum products,
there are many more modifications to be made. One of these involves the gene cpcB
that codes for a protein, called phycocyanin, that captures light energy in the
photosynthetic process. Phycocyanin efficiently captures solar energy, but also
prevents cyanobacterial cultures from growing to high densities because the cells
closest to the surface absorb most of the sunlight and prevent the cells beneath from
receiving sufficient light. This is known as ‘overshadowing.’ As a result cultures are not
able to grow to high densities with high bioproduct yields. My goal is to inactivate the
cpcB gene by inserting an antibiotic resistance gene into it. This will prevent
phycocyanin from being made, which in turn should prevent the overshadowing effect
and result in higher isoprene yields. I have used genetic engineering methods to
assemble a DNA construct that carries the inactivated cpcB gene and replicates as a
plasmid (extra-chromosomal element) in a bacterial host. This inactivated gene will be
introduced into Synechococcus to generate cyanobacteria that can be grown to higher
densities with increased isoprene yields. Producing higher amounts of isoprene is one
of the first steps to creating a marketable replacement for petroleum products.
Introduction
The use of cyanobacteria to produce bio-molecules is a viable option to replacing
petrochemical products. Cyanobacteria are oxygenic photosynthetic prokaryotes
(Collins et. al, 2012), and our group has engineered Synechococcus cyanobacteria to
produce isoprene via the methyerythritol phosphate (MEP) pathway (Figure 1). Part of
the photosynthetic process requires phycobilisome (PBS) light-harvesting protein
complexes that efficiently capture solar energy (Figure 2). Cell density and bioproduct
formation in cyanobacterial cultures is limited by an ‘overshadowing’ effect. Cells
growing at the surface of a culture capture most of the solar energy and overshadow
cells growing beneath them. Our solution to this problem is to inactivate the cpcB gene
for the major phycocyanin component of the PBS. By inactivating the cpcB gene,
cultures of Synechococcus 7002 will produce smaller PBS and can be grown to higher
densities, which in turn should allow for higher isoprene production per culture
volume. Genetic engineering methods were used to develop a DNA construct that will
be used to inactivate the cpcB gene for phycocyanin in Synechococcus.
Decreased light-harvesting for increased cell
density and isoprene yield
Figure 2. Generic cyanobacterial phycobilisome
light-harvesting complex. Energy is funneled from
phycoerythrin (PE) to phcocyanin (PC) to allophycocyanin
(APC) to the reaction center. Synechococcus 7002 does
not have PE. (http://www.genome.ad.jp/kegg/). Deletion
of the cpcB gene will eliminate the PC component.
Gel image of DNA fragments used in Gibson
Assembly of ∆cpcB gene construct
Carbon flow & MEP pathway for isoprene synthesis in
cyanobacteria
Figure 3: Gel image of fragments to be used
for Gibon Assembly. Lane 1: 1kB Ladder, Lane 2:
Upstream cpcB gene, 676bp, Lane 3: Downstream
cpcB gen.e 588bp, Lane 4: PUC57 plasmid replicon,
2400bp, Lane5: CmR resistance gene, 1500bp.
Figure 1. Methyl-D-erythritol-4-phosphate
(MEP) pathway. CO2 captured by photosynthesis
leads via the MEP pathway to isopentenyl
diphosphate (IPP) and dimethylallyl-diphosphate
(DMAPP). Abbreviations for metabolites in the MEP
pathway are shown in black, and abbreviations of
enzyme names are in red. Optimized IspS and IDI
genes have been introduced into Synechococcus to
obtain isoprene production.
(Toivo Kallas, UWO, AABT, Algal Bioproducts-Biofuels, 6-2014)
Methods and Materials
The first step was to extract chromosomal DNA from Synechococcus 7002. This
DNA, from the areas upstream and downstream of the cpcB gene, was then
amplified by polymerase chain reaction (PCR) and the products used for Gibson
Assembly, along with DNA from two other plasmids. The first plasmid was
pOSH1108, and a region from plasmid pUC57 was used as a backbone (‘replicon’)
for the construct. The second plasmid was pRL409, which is where the
chloramphenicol resistance gene was acquired from. Four total fragments were
used to construct the ΔcpcB plasmid. The four fragments consisted of the
following: Upstream cpcB gene, downstream cpcB gene, pUC57 plasmid backbone,
and chloramphenicol resistance (CmR) gene. The next step was to introduce the
assembled plasmid into E. coli bacteria to verify that the DNA construct carrying
the inactivated cpcB gene (named ∆cpcB) had been correctly assembled. Once
growth of E. coli was observed in the presence of chloramphenicol, this plasmid
DNA was extracted from the cells. The plasmid DNA was characterized by
restriction digestion and PCR. Once confirmed, PCR was used to amplify the region
of the plasmid containing the upstream and downstream regions of cpcB gene, and
the chloramphenicol resistance gene. This DNA fragment containing these three
regions was introduced into Synechococcus 7002 by genetic ‘transformation,’ and
cells were plated onto growth medium containing chloramphenical to select for
cyanobacteria that had integrated the inactivated cpcB gene into their
chromosome.
Upstream
cpcB gene
pUC57 Backbone
∆cpcB Inactivation Plasmid
CmR gene
Downstream
cpcB gene
Figure 4. Constructed ∆cpcB inactivation plasmid.
Important DNA segments are as follows, Blue: upstream
and downstream portions of cpcB from Synechococcus
7002, Pink: Chloramphenicol resistance gene, Green:
pUC57 plasmid backbone.
Growth of E. coli transformants in
liquid medium
Results
Chromosomal DNA from Synechococcus sp. PCC 7002 was amplified by means of PCR,
along with regions from two other plasmids to obtain the pUC57 plasmid backbone
as well as the chloramphenicol resistance gene. The results from the PCR are shown
in Figure 3. These data show that the correct size fragments were attained. These
DNA fragments were then assembled in a ‘Gibson Assembly’ reaction to form a
plasmid that carries the inactivated cpcB gene, as shown in Figure 4. The plasmid
pictured, the ∆cpcB inactivation plasmid, contains four fragments: upstream and
downstream segments that correspond to the regions surrounding the cpcB gene in
Synechoccocus 7002 chromosome, the pUC57 backbone, as well as the
chloramphenicol resistance gene (CmR). To verify that the correct plasmid was
assembled it was introduced to E. coli. Figure 5 shows E. coli colonies (transformants)
that were selected for growth on medium containing chloramphenicol (Cm), which
allows only the growth of cells that have taken up the ∆cpcB inactivation plasmid.
Several of these colonies were inoculated into liquid medium containing Cm in
preparation for plasmid analysis and introduction of the ∆cpcB DNA into
Synechococcus 7002. As seen in Figure 6, tube 2 showed dense, overnight growth of
one of the E. coli transformants in the presence of chloramphenicol, suggesting that
these cells contain the properly constructed ∆cpcB ‘knockout’ plasmid. PCR and DNA
sequencing will be done to confirm the correct assembly of the ∆cpcB plasmid. DNA
from this plasmid was introduced into Synechococcus 7002 with selection for growth
on Cm to obtain mutant strains that carry the inactivated ∆cpcB gene. Figure 7 shows
growth of possible Synechococcus7002 ∆CpcB transformants in the presence of
chloramphenicol. This will need to be verified by single colony isolation and PCR
analysis to show integration of the ∆cpcB DNA into the genome with replacement of
the native cpcB gene for the phycocyanin light-harvesting protein.
Figure 5. Transformed E. coli growing on agar
medium containing chloramphenicol. These cells
carry the ∆cpcB inactivation plasmid with its CmR gene.
Marked colonies were selected to be transferred into
liquid culture for plasmid extraction and analysis.
Conclusions
The growth of Synechococcus 7002 transformants (cells that
have taken up DNA) in the presence of chloramphenicol
suggests that the cpcB gene may have been inactivated in
these cyanobacteria. However, this will need to be confirmed
by single colony isolation and tests that include PCR
amplification and restriction enzyme digests. Absorbance
spectra of cell extracts will show whether the ∆cpcB deletion
results in loss of phyocyanin from phycobilisome lightharvesting complexes. Finally, Synechococcus 7002 will be
grown in liquid culture to determine whether the ∆CpcB
deletion prevents self-shading, allows cultures to grow to a
higher density, and results in higher isoprene production
yields per culture volume.
Figure 6. Growth of putative E. coli ∆cpcB
transformants in liquid medium with Cm. Tube
2 showed dense overnight growth. DNA from this
suspension was used to transform Synechococcus
cyanobacteria.
Possible Synechoccocus 7002 ∆cpcB
transformants
Acknowledgements
I would like to express my appreciation to Dr. Kallas for the
opportunity to work in his lab. Also I would like to thank
Andrea Felton for teaching me and offering guidance on the
project. My gratitude for the opportunities offered by the
University of Wisconsin Oshkosh McNair Program are endless
and I would like to thank Mary Seaman and Kelly Hanson for
their continued assistance. The UW Oshkosh McNair Scholars
Program is 100% funded through a TRIO grant from the United
States Department of Education PR/Award Number
P217A120210. For 2013/2014, the UW Oshkosh McNair
Scholars Program will receive $208,494 each year in federal
funds.
References
Figure 7. Growth of putative Synechococcus
7002 ∆cpcB transformants in the presence
of chloramphenicol. The green patch on the
left of the plate may contain possible ∆CpcB
transformants, but individual transformant
colonies have not yet been obtained.
Collins, Aaron M.. "Photosynthetic Pigment Localization and
Thylakoid Membrane Morphology Are Altered in Synechocystis 6803
Phycobilisome Mutants." Plant Physiology 158: 1600-1609.