CALIFORNIA STATE UNIVERSITY NORTHRIDGE
Characterization of Neutral Lipid Production of Nostoc punctiforme
A thesis submitted in partial fulfillment of the requirements
for the degree of Master of Science
in Biology
By
Anantha Peramuna
August 2013
The thesis of Anantha Peramuna is approved by:
______________________________________
Sean Murray, PhD
__________________
Date
______________________________________
Chhandak Basu, PhD
__________________
Date
______________________________________
Michael L. Summers, PhD, Chair
__________________
Date
California State University, Northridge
ii
Acknowledgement
I especially would like to thank Dr. Summers for giving me the opportunity to
work in this lab and for being a great mentor and a friend. Thank you to my
committee members, Dr. Chhandak Basu and Dr. Sean Murray. Also, thank you
to my fellow lab mates who made our work environment fun and friendly.
This project was made possible by funding provided by CSUPERB and a NIH
SCORE Grant1SC1GM093998 to MLS.
iii
Table of Content
Signature Page
ii
Acknowledgments
iii
List of Figures
vi
Abstract
viii
Introduction
1
Cyanobacterial Photosynthesis
2
Cyanobacterial Inclusion Bodies
3
Biofuel Production in Photosynthetic Microorganisms
5
Neutral Lipid Biosynthesis
6
Altering Metabolic Pathways to Enhance Neutral Lipid Production
11
Visualizing Neutral Lipids
12
Methods Used to Study Lipids
12
Materials and Methods
16
Bacterial strains and growth conditions
16
Neutral Lipid Visualization
16
Identification of Proteins Involved in Neutral Lipid Droplet Formation
17
Selecting for a Neutral Lipid Enriched Culture Using Differential
18
Density Centrifugation
18
Neutral Lipid Formation under Various Growth Conditions
19
Neutral Lipid Identification
19
iv
Determination of Fatty Acid Composition
20
Metabolic Engineering
22
Results
32
Discussion
63
Citation
67
Appendix
70
v
LIST OF FIGURES
Figure 1- Chemical reaction showing conversion of uncharged (neutral) lipids
into biodiesel
5
Figure 2- A. Known pathways for TAG biosynthesis. B. Formation of ester bond
between fatty acyl CoA and the DAG, catalyzed by DGAT
8
Figure 3- Proposed model for neutral lipid droplet formation in eukaryotes
9
Figure 4- Proposed mechanism for neutral lipid formation in prokaryotes
9
Figure 5- Separation of proteins on a protein gel
32
Figure 6- Separating the cells with highest lipid content
33
Figure 7- Stained with Bodipy and visualized using maximum projection on
confocal microscope
34
Figure 8- Measuring the area of the filaments using Image J
34
Figure 9- Measuring the area of the lipid globules using Image J
34
Figure 10- Neutral lipid staining with the fluorescent dye Bodipy and
Visualization using laser confocal microscopy
35
Figure 11- Quantification of stained area for 50 cells (grown with
MOPS/ammonia/nitrate) with ImageJ
35
Figure 12- Relative neutral lipid area relative to the cell size
37
Figure 13- Neutral lipid separation on TLC using different extraction methods 38
Figure 14- Neutral lipid separation on TLC using different solvent systems
40
Figure 15- Neutral lipid separation on TLC using different solvent systems
41
Figure 16- Fatty acid methyl esters of neutral lipids
43
Figure 17- GC-MS results of neutral lipids extracted from N. punctiforme
wild type cells grown in MOPS/Ammonia (MA)
44
Figure 18- GC-MS results of neutral lipids extracted from N. punctiforme
wild type cells grown in MOPS/Ammonia (MA)
45
Figure 19- GC-MS results of neutral lipids extracted from N. punctiforme
wild type cells grown in MOPS/Ammonia (MA)
46
vi
Figure 20- GC-MS results of neutral lipids extracted from N. punctiforme
wild type cells grown in MOPS/Ammonia (MA)
47
Figure 21- GC-MS results of neutral lipids extracted from N. punctiforme
wild type cells grown in MOPS/Ammonia (MA)
48
Figure 22- GC-MS results of neutral lipids extracted from N. punctiforme
wild type cells grown in MOPS/Nitrate (MN)
49
Figure 23- GC-MS results of neutral lipids extracted from N. punctiforme
wild type cells grown in MOPS/Nitrate (MN)
50
Figure 24- GC-MS results of neutral lipids extracted from N. punctiforme
wild type cells grown in MOPS/Nitrate (MN)
51
Figure 25- GC-MS results of neutral lipids extracted from N. punctiforme
wild type cells grown in MOPS/Nitrate (MN)
52
Figure 26- GC-MS results of neutral lipids extracted from N. punctiforme
wild type cells grown in MOPS/Nitrate (MN)
53
Figure 27- GC-MS results of neutral lipids extracted from N. punctiforme
wild type cells grown in MOPS/Nitrate/Fructose (MNF)
54
Figure 28- GC-MS results of neutral lipids extracted from N. punctiforme
wild type cells grown in MOPS/Nitrate/Fructose (MNF)
55
Figure 29- GC-MS results of neutral lipids extracted from N. punctiforme
wild type cells grown in MOPS/Nitrate/Fructose (MNF)
56
Figure 30- GC-MS results of neutral lipids extracted from N. punctiforme
wild type cells grown in MOPS/Nitrate/Fructose (MNF)
57
Figure 31- GC-MS results of neutral lipids extracted from N. punctiforme
wild type cells grown in MOPS/Nitrate/Fructose (MNF)
58
Figure 32- Concentration of fatty acid chains in wild type cells grown in
different growth conditions
59
Figure 33- Confirming the glycogen mutant plasmid construct by colony PCR
Figure 34- Confirming the double recombinant glycogen mutant
62
Figure 35- Confirming the cyanophycin mutant plasmid construct by
plasmid digest
62
vii
Abstract
Characterization of Neutral Lipid Production of Nostoc punctiforme
By
Anantha Peramuna
Master of Science in Biology
Nostoc punctiforme is a member of the cyanobacteria that forms filaments
of cells and is capable of forming lipid droplets in the cytoplasm. References to
lipid droplets in cyanobacteria are scattered throughout the cyanobacterial
literature, and are mostly limited to merely describing their presence in electron
micrographs. The goal of this work is characterize neutral lipid production in the
cyanobacterium Nostoc punctiforme, and to alter metabolic pathways that might
favor its production.
Neutral lipids of Nostoc punctiforme were characterized by extraction,
separation using appropriate solvent systems unique for separation of
diacylglycerol (DAG) and triacylglycerol (TAG) by thin layer chromatography,
and visualized using iodine. Results indicate that Nostoc punctiforme produces
both DAG and TAG. Neutral lipids of Nostoc punctiforme were also turned in to
fatty acid methyl esters (FAME) and analyzed using gas chromatography and
mass spectrometry (GC-MS). GC-MS results suggest that neutral lipids of Nostoc
punctiforme are mainly composed of C16, C16:1, C18, C18:2 and C18:3 fatty
acid chains. Wild type cells grown in different growth conditions suggest that the
composition of the neutral fatty acids could change with the growth media.
Whole-cell staining with BODIPY and visualization by laser confocal
microscopy indicated neutral lipids were contained in lipid globules within cells.
The quantity of neutral lipid globules increases with the age and the growth
condition of the culture. Log phase has considerably fewer and smaller neutral
lipid globules compared to the stationary phase. Cells grown in
Mops/Nitrate/Fructose had the highest concentration of neutral lipids per cell area
in log phase and cells grown in Mops/Nitrate had the highest concentration of
neutral lipids per cell area in stationary phase cultures.
A strain that overproduces neutral lipids was developed using buoyancy as
selection. Sonicated filaments were layered on a Percoll gradient and centrifuged.
Upper fractions that contain the most buoyant filaments were selected, re-grown
to early stationary phase, and subjected to additional rounds of selection. After
eight rounds of Percoll selection, quantification of lipid area per cell from digital
images indicated that the selected strain produced twice as many neutral lipids as
the wild type strain under similar growth conditions. Individual clones arising
viii
from this culture are being screened to identify those with maximal lipid globule
production.
To see if neutral lipid production could be further increased by genetic
modification, gene deletion plasmids were been constructed to block non-essential
carbon storage pathways. A glucose-1-phosphate adenylyltransferase deletion
plasmid was created by PCR mediated ligation and conjugated into Nostoc
punctiforme to create a mutant unable to synthesize glycogen. A second mutation
in cyanophycin synthatase, expected to block cyanophycin production (a nonribosomally produced amino acid polymer used for carbon and nitrogen storage in
cyanobacteria) is also in progress.
ix
Introduction
Introduction to Cyanobacteria
Cyanobacteria are a highly diverse group of organisms ranging from
single cells to filaments, some of which are large enough to be visible without
using a microscope. Historically, cyanobacteria were termed as blue green algae
and classified botanically (Rippka et al., 1979). Among various types of bacteria,
cyanobacteria exhibit an unusually wide range of morphologies and classified into
five groups focusing on their morphology and development (Rippka et al., 1979).
Group 1 (Chroococcales) is made out of solitary and colonial unicellular
cyanobacteria, for example, Synechococcus and Gloeocapsa. Group 2
(Pleurocapsales) ranges from unicellular to pseudo-filamentous, thallus-forming
cyanobacteria, with cells capable of cell division in multiple planes as well as
binary fission. Group 3 (Oscillatoriales) includes filamentous cyanobacteria that
do not differentiate. Group 4 (Nostocales) comprises of filaments marked by cell
differentiation
to
producing
spore
like
akinetes
and
nitrogen
fixing
heterocysts. Nostoc punctiforme is an example of this group that was used in this
work. It has a fully sequenced genome and the capacity to form all of the cell
types mentioned above. Group 5 (Stigonematales) consist of cell-differentiating
cyanobacteria with more complex multicellular organization (Herrero et al., 2008).
These groups have colonized most of earth’s environments due to their
photoautotrophic lifestyle and minimal requirements.
One aspect that makes certain species of cyanobacteria so well adapted to
colonizing diverse habitats is the ability to fix nitrogen. Nitrogen fixation in
1
cyanobacteria occurs in free-living cells and also when they are in symbiotic
association with plants, fungi, and various eukaryotic algae. The heterocyst is the
specialized cell type where nitrogen fixation takes place in the Nostocales.
Cyanobacteria are also capable of using different inorganic and organic nitrogen
compounds, and when present, they typically inhibit heterocyst formation and
therefore nitrogen fixation (Herrero et al., 2008)
Cyanobacterial Photosynthesis
Cyanobacteria are considered Gram negative, meaning their envelope
contains an outer membrane, a peptidoglycan layer and a plasma membrane. They
contain a unique internal membrane system termed the thylakoid membrane
where photosynthesis takes place (Norling et al., 1998). The chlorophyll
containing thylakoid membrane is responsible for oxygenic photosynthesis. The
cyanobacterial
photosynthetic
apparatus
consists
of
two
photosystems;
photosystem 1(PS 1) and photosystem 2 (PS 2) similar to those found in plants.
They work together as a series capturing light and transferring electrons in the
thylakoid membrane. Photosystems mostly contain chlorophyll to capture light,
but the problem with chlorophyll is that it does not capture light across the entire
spectrum. For an example chlorophyll a only absorbs light around 430-440nm and
again at 670nm, this could be inefficient as it leaves a huge range of light in the
spectrum as unusable. To solve this problem cyanobacteria utilize an antenna
complex called the phycobilisome that sits on the cytoplasmic side of the
thylakoid membrane. Phycobilisomes are mostly present in PS 2 and contain
three
predominant
phycobiliproteins;
2
phycoerythrin,
phycocyanin
and
allophycocyanin. These phycobilisomes are capable of capturing light between
450-660 nm and transferring energy to the photosystems, making the
cyanobacterial photosynthetic apparatus more efficient (Herrero et al., 2008).
Although an ancient cyanobacterium is thought to be the ancestor of the
chloroplasts found in today’s plants, studies done on thylakoid membranes of both
cyanobacteria and chloroplasts have revealed many exclusive functions. In
chloroplasts, thylakoid membranes lack phycobilisomes and develop from
vesicles that are formed in the chloroplast’s inner envelope membrane, making
the thylakoid a completely separate internal membrane system. In cyanobacteria,
in contrast, it is not fully known if the internal thylakoid membrane is fully
separated from the cytoplasmic membrane or if it remains attached (Fuhrmann et
al., 2009). Since thylakoid membrane formation is positively correlated with
photosynthetic rate, the highest density of lipids in cyanobacterial biomass occurs
together with the highest biomass (Sheng et al., 2010).
Cyanobacterial Inclusion Bodies
Cyanobacteria have a few cytoplasmic inclusions; one of them is the
carboxysome. Carboxysomes are present in all vegetative cyanobacterial cells.
They contain ribulose-bisphosphate carboxylase, which is a key enzyme in fixing
carbon dioxide via reductive pentose phosphate (Calvin Benson) pathway.
Carboxysomes are absent from fully differentiated heterocyst since they are nondividing terminally differentiated cells that do not fix carbon dioxide.
3
Glycogen granules are another type of inclusion. Glycogen is composed of
poly glucose and is the carbohydrate reserve material in cyanobacteria. ADPglucose pyrophosphorylase is the enzyme involved in cyanobacterial glycogen
synthesis, and it is encoded by the agp gene. Although glycogen granules are
rarely present in exponential growth, they are abundant in stationary phase..
Another noticeable inclusion body is the cyanophycin granule.
Cyanophycin is specific to cyanobacteria and is made of long polymeric
molecules of L-aspartic acid and L-arginine. An enzyme called cyanophycin
synthetase incorporates L-aspartic and L-arginine into cyanophycin with the help
of an ATP resulting in a 1:1 stoichiometry of aspartic to arginin. The production
of this polymer does not require any ribosomes. The purpose of cyanophycin is to
act as a nitrogen and carbon reserve. As with glycogen, cyanophycin granules are
more visible in cells that are approaching stationary phase (Zeigler,1998).
Another important inclusion that is present in cyanobacteria is the lipid droplet.
They appear as electron-opaque, circular, small droplets that are variable in size.
These droplets are frequently seen among the thylakoids (Packer, 1988). The
exact enzymes involved in formation of this lipid droplet polymer are unknown in
microorganisms (Waltermann et al., 2005). From a metabolic engineering
perspective, blocking the synthesis of the other two types of storage granules
could force more carbon incorporation into lipid droplets to produce fuel (Sheng,
2010).
4
Biofuel Production in Photosynthetic Microorganisms
Biofuel is defined as esters derived from renewable biological sources in
the form of alcohols, oils or fats. Demand for clean and sustainable biofuel has
gained a lot of support recently due to the energy demand and climate change.
Figure 1- Chemical reaction showing conversion of uncharged (neutral) lipids into
biodiesel (Angermayr, 2009)
Biofuel production in algae, plants, and bacteria can be thought of as a two
step process. In the first step solar energy is used to incorporate CO2 into organic
molecules. In the next step small portion of these molecules make alcohols or
fatty acid esters (Angermayr, 2009). Since sunlight and CO2 is abundant in nature,
biofuel production using photosynthesis seems sustainable (Kim, 2010). Also
since these photosynthetic organisms uses CO2 as a source for growth there would
be no net gain of CO2 in the atmosphere, unlike burning fossil fuel
(Balasubramanian, 2010).
Biofuel production using algae and cyanobacteria is much more efficient
than using plants; photosynthetic efficiency of cyanobacteria is ten times higher
than plants and not having to support production of tissues such as roots and
stems further increases their productive output (Ducat, 2012). Microalgae could
5
produce up to 100,000 L of oil/ha/year which is 16-70 times more than the yield
of oil from plants (Balasubramanian, 2010). Also, since they don’t compete for
arable land, they don’t cause food shortage problems (Dismukes et al.,
2008). Currently, algae have been the most popular choice for microbial biofuel
production because they are capable of producing a significant amount of
triacylglycerol (TAG) as a storage lipid. Conversion of TAG into biofuel requires
the release of the fatty acids from the glycerol backbone (Fig.1). However,
producing this kind of lipid at a commercial scale could be costly and complicated
since microalgae require certain stresses such as nitrogen starvation to create
TAG (Hu et al., 2008).
Neutral Lipid Biosynthesis
Lipids are divided into two main groups based on their chemical
characteristics, polar lipids and neutral lipids. Neutral lipids are the least polar of
the lipids containing waxes, isoprenoids, monoglycerides, diglycerides and
triglycerides.
Polar
lipids
are
the
charged
lipids
of
phospholipids
(phosphatidylinositol, phosphatidylcholine etc) and glycolipids (monogalactosyl
diglyceride).The choice of biodiesel has been triacylglycerol since it yields more
energy. The composition of the algal lipids could vary depending on the species,
nutrients and growth conditions. Typically, microorganisms tend to increase the
production of triacylglycerol due to environmental stresses and nutrient starvation.
When faced with stressful conditions they increase their TAG production by
decreasing the growth rate (Greenwell, 2010).
6
There are two known major pathways for TAG biosynthesis in eukaryotic
cells: the glycerol phosphate (GP) pathway and monoacylglycerol (MG) pathway
(Fig. 2A). The GP pathway is present in most cells, but the MG pathway is
known to be present in only specialized cells such as hepatocytes, adipocytes and
enterocytes. At the end of both pathways diacylglycerol (DAG) and a fatty acylCoA is covalently bonded to form TAG. This reaction is catalyzed by the
diacylglycerol acyltrasnferase (DGAT) enzyme (Fig. 2B) (Yen, 2008)
…
.
Figure 2- A. Known pathways for TAG biosynthesis. B. Formation of ester bond
between fatty acyl CoA and the DAG, catalyzed by DGAT (Yen, 2008).
7
TAG biosynthesis takes place in membranes; however the exact
mechanism of how TAG is incorporated into neutral lipid droplets is unknown.
Two of mechanisms have been proposed for eukaryotic systems, both involving
either an apolipoprotein or some kind of protein(s) striping neutral lipids off
membranes and helping them to navigate through water to the lipid globule (Fig.
3) (Yen, 2008).
Figure 3- Proposed model for neutral lipid droplet formation in eukaryotes (Yen,
2008).
The model for neutral lipid droplet formation in prokaryotes comes from
Acinetobacter calcoaceticus ADP1 and R. opacus PD630 (Fig. 4). According to
this mechanism, wax ester synthase/acyl-CoA:diacylglycerol acyltransferase
(WS/DGAT) attaches to the cytoplasm membrane and synthesizes small lipid
droplets (SLD), this oily layer gets coated by a monolayer of phospholipids. SLDs
start coalescing and forms lipid droplets which then bud out from the membrane
into the cytoplasm (Waltermann et al., 2005).
8
Figure 4- Proposed mechanism for neutral lipid formation in prokaryotes
(Waltermann et al., 2005)
A cyanobacterial cell contains several different types of lipids. TAG is
known to be the major kind of neutral lipid (Singh, 2002). They also have four
different kind of other major lipids distinguishable by their head-groups;
monogalactosyl diacylglycerol (MGDG), monoglucosyl diacylglycerol (GlcDG),
digalactosyl diacylglycerol (DGDG), sulfoquinovosyl diacylglycerol (SQDG) and
phosphatidylglycerol. Since cyanobacterial fatty acids contain un-branched chains
containing 14, 16 or 18 carbons (Packer, 2008), it could be a potential alternate
diesel fuel (Singh, 2002). Length of the carbon chains in cyanobacteria could
differ based on the species. For an example, Anacystis nidulans (non-nitrogen
fixing) lipids are composed of 16:0 and 16:1 carbons while Anabaena cylindrica
(nitrogen fixing) contains predominantly 18:2 and 18:3 carbon chains (Sall, 1990).
The saturation of the fatty acid in cyanobacteria could change based on their
environment. Cyanobacteria that live under reduced oxygen conditions tend to
lack polyunsaturated fatty acids (Oren, 1985).
These lipids are associated with membranes and lipid globules, when
present. Although all of these lipid sources could be used as potential biofuel
9
sources, a problem rise when using membrane lipids since a lot of pathways are
involved in making these lipids. As a result it’s hard to calculate and quantify the
output of a particular pathway, since many of these lipids have charged or polar
head groups, it could be hard to purify them as well. On the other hand lipid
globules, also known as lipid bodies, seems to have a genetically or
physiologically (by controlling the growth conditions) inducible expression
(Wang, 2009). And since they are uncharged (neutral lipids as will be shown in
this work), purifying them could be easier than the membrane lipids (Cooper,
2009).
Although lipid globules are known to be present in cyanobacteria, little is
known about their formation or function. It has been hypothesized that these lipid
globules form due to starvation or photo-oxidative damage. A possible
explanation for photo-oxidation damage and lipid globule formation comes from
plants. Plants, algae and cyanobacteria have a family of proteins mostly found in
lipid globules called plastoglobulins (PGL). These globulins are scattered
throughout the stroma and are known to initiate or in some way assist lipid
globule formation in plants. Lipid globules extracted from Arabidopsis thaliana
chloroplasts have confirmed the presence of plastoglobulins. PGL is also known
to play a role in protecting the photosynthetic apparatus from photo-oxidative
damage. An emerging concept for the role of PGL states that lipid globules serve
as a deposit site for potentially toxic free fatty acids, carotenoids, components of
the electron transport chain and other lipophilic compounds that become unbound
10
by the photo oxidative damage. These lipid globules later may be used as raw
material for thylakoid repair and remodeling (Cunningham, 2010).
Altering Metabolic Pathways to Enhance Neutral Lipid Production
Cyanobacteria that have a facile genetic system (such as N. punctiforme)
are attractive candidates for biofuel production (Ducat, 2012). Based upon studies
in algae, lipid globule production in cyanobacteria could be enhanced by
eliminating alternate storage polymers such as glycogen or cyanophycin. This is
supported by the observation that the algae Chlamydomonas reinhardtii produced
twice as many lipid bodies when the gene encoding ADP glucose phosphorylase
(AGPase) was deleted. This enzyme makes ADP-glucose from glucose-1phosphate and ATP. ADP-glucose serves as the glucosyl donor in α-glucan
synthesis. This result suggested that carbons that normally go into starch
formation were routed to the fatty acid cycle (Wang, 2009). In cyanobacteria, the
agp gene encodes AGPase. Deletion of agp in Synechocystis sp. PCC 6803
completely blocked the glycogen biosynthesis, though neutral lipid formation in
these mutants wasn’t studied (Miao, 2002).
Another approach to find genes involved in lipid production is the use of
proteomics. Moellering (2010) found 259 proteins associated with neutral lipiddroplet enriched fractions from C. reinhardtii. One of the important proteins was
a major lipid droplet protein (MLDP). When MLDP was inactivated in C.
reinhardtii, the size of the lipid droplet increased, however the total levels of
TAG in the droplets did not change (Moellering, 2010).
11
Visualizing Neutral Lipids
Lipid globules formed inside cyanobacteria and algae could be visualized
using BODIPY 505/515 (4,4-difluoro-1,3,5,7- tetramethyl-4-bora-3a,4a-diaza-sindacene). BODIPY 505/515 is known to specifically stain neutral lipids, and has
been used to stain lipid-containing yolk platelets in live zebra fish embryos as
well as lipid-containing vesicles in immortalized human hepatocytes (Cooper,
2010). When live algal cells are stained with BODIPY 505/515, intracellular oilcontaining organelles stain within minutes. BODIPY 505/515 has a high oil/water
partition coefficient, which aids the dye in easily crossing the cell and the
organelle membranes. It accumulates in lipid-rich intracellular compartments by a
diffusion-trap mechanism. Stained lipid bodies can be visualized by wide-field
epifluorescene microscopy or confocal microscopy. Most importantly for this
work, BODIPY 505/515 is non-toxic; algal cells remained viable after staining
and could be used for re-inoculation after being passed through a FACS
instrument (Cooper, 2010). Another popular stain that has been used throughout
the years is Nile red. Nile red has been used to stain lipid content from
microorganisms to animals (Chen, 2009). It has been documented that
permeability of Nile red varies among algal species. It is recommended to study
the algal fluorescence over 30-40 minutes for the maximum intensity once the
subjects are being stained with Nile red. Nile red also have a broader emission
spectrum (515-530/525-605) compared to Bodipy and might not be as useful
when used with confocal microscopy (Cooper, 2010).
12
Methods Used to Study Lipids
Total lipids of a cell can be extracted using the method of Bligh and Dyer
that involves solubilization of the lipids in a mixture of chloroform, water and
methanol. According to this method all the lipids (neutral, polar, and charged) end
up in the chloroform layer and the non-lipids ends up in the methanol layer (Bligh,
1959). Since, Bligh and Dyer method also extracts charged lipids and may not
fully extract all neutral lipids, Hutchins et al., have proposed a new extraction
method to extract only neutral lipids. According to their method, neutral lipids are
extracted by mixing the lysed cells with isooctane/ethyl acetate [75:25 (v/v)] and
extracting the organic layer after centrifuging for five minutes at 1000 x g. To
increase the yield, neutral lipids are being re- extracted with the same solvent
system (Hutchins, 2008).
Thin layer chromatography (TLC) can be used to separate large molecules
such as lipids. This is typically done on a sheet of glass coated with an absorbent
such as silica (stationary phase). Separation of the compounds are archived by
spotting a mixture on the stationary phase and using a solvent (mobile phase) to
carry the mixture via capillary action (Vogel, 1996). The mixture is separated by
difference in the rate of travel due to their affinity to the stationary phase, and the
differences in their solubility in the solvent mixture. TAG, DAG, and Neutral free
fatty acids can be identified on a TLC plate with proper standards. These
separated molecules could be colorless and may need staining methods to be
visualized. One way to visualize these colorless spots is to use iodine vapor.
13
Iodine vapor has a higher affinity to aromatic and unsaturated compounds and
would bind with the colorless molecules to give a dark brown color (Fair, 2008).
The composition of the fatty acid hydrocarbons in TAG and DAG can be
identified by gas chromatography (GC). Fatty acids from TAG and DAG are first
turned into fatty acid methyl esters (FAME) before been used for GC analysis.
This is done in two steps where first, the glycerol backbone is removed from the
fatty acid esters (saponification), followed by methyl esterification where
methoxide ion is attached to the fatty acid esters (Ma, 1999). FAME samples are
then injected into a GC where the FAME gets vaporized and carried by an inert or
an unreactive gas such as helium or nitrogen (mobile phase) through a column
that is coated with a stationary phase. Gaseous molecules interact with the wall of
the column and elute at different times (retention time). Composition of the fatty
acids could be determined by comparing known retention times of the FAME
standards to that of the unknown (Harris, 1999).
Overview
Reference to lipid droplets in cyanobacteria are scattered throughout the
cyanobacterial literature, and are mostly limited to merely describing their
presence in electron micrographs (Packer, 1988), (Van De Meene, 2006) and
(Cunningham, 2010). This work 1) documents their presence in the
cyanobacterium Nostoc punctiforme, 2) tries to identify potential lipid-associated
proteins that may be involved in lipid globule formation or function. 3) describes
a method for their visualization and quantifies their expression under different
14
modes of growth, 4) characterizes neutral lipids and their fatty acid constituents,
and This work also initiates experiments to alter the metabolic pathways of N.
punctiforme hypothesized to result in enhanced neutral lipid production.
15
Materials and Methods
Strains and Growth Conditions
Nostoc punctiforme- cells were grown in the standard minimal salt
medium of Allan and Arnon (1955), diluted four folds and supplemented with one
of the following media (pH 7.8). 5 mM MOPS /5 mM Ammonia (MA) , 5 mM
MOPS /Nitrate [2.5 mM NaNO3 and 2.5 mM KNO3 (MN)], 5 mM MOPS only, 5
mM MOPS /Nitrate (2.5 mM NaNO3 and 2.5 mM KNO3 )/ 50 mM Fructose
(MNF). The cultures were shaken at 125 rpm and exposed to 15.75 µmol s-1 m-2
lighting in room temperature. E. coli- cells were grown in Luria Bertani (LB)
broth that contains 1% tryptone, 0.5% yeast extract and 0.5% NaCl titrated to pH
7.2 with 30 μg/ml Km. The cells in LB were grown in test tubes using a rolling
incubator at 370C.
Neutral Lipid Visualization
Neutral Lipids were stained using BODIPY 505/515 (Invitrogen Molecular
Probes, Carlasbad, CA). A 5mM stock solution was made using DMSO (vol/vol).
Cells were visualized with a final BODIPY concentration of 0.5µM. 10µl of
0.5µM BODIPY was added to 0.5µg Chla/100µl of cells.
Epifluorescence micrographs were obtained using 8µl of BODIPY stained
cells loaded on a 1% agarose gel pad under a cover slip and visualize using
confocal microscopy (Leica LAS AF ). Cells were excited at 488nm (20% laser
power) and visualize using a window of 510-530nm. Size of the filaments was
increased to a desirable size by using 1024 X 512 pixel frames and increasing the
gain (3 - 4.5). Z stacks were performed on each filament with the default setting
except the line averaging set at 4. Z-stacks were cropped just to include the planes
16
of the cells and a maximum projection was done to bring all the individual Zstacks together.
The percentage of neutral lipids present per cell was measured using the
software Image J. Area of the neutral lipids were measured by selecting the area
of neutral lipids using the freehand selection function and measuring the area of
the pixel within the chosen area. Area of the cells was determined the same way.
Percentage of the neutral lipids per cell was determined by dividing the pixel area
of the neutral lipids by pixel area of the cell and multiplying by 100.
Identification of Proteins Involved in Neutral Lipid Droplet Formation
Cells were suspended in lysis buffer (0.5M Sucrose, 50mM Tris buffer (pH
7.8), 5mM MgCl2, 5mM KCl, 1X Protease inhibitor, 10mg/ml lysozyme) using
25ml per 3g of wet weight stationary phase cells. The suspension was incubated
for two hours at room temperature, inverting the tubes, occasionally, followed by
lysis using a single pass through a cold French press at 18,000 psi. The lysed cells
were added to an ultracentrifuge tube, and overlayed with over lay buffer (50mM
Tris buffer (pH 7.8), 5mM MgCl2, 5mM KCl, 1X Protease inhibitor), and
centrifuged at 135,000 x g at 4oC for 2 hours with acceleration set at 5 and
deceleration at 5 (SORVALL Discovery 90 centrifuge) using an AH 629 bucket
rotor.
The top yellow oily layer of the centrifuged sample was collected with a Jshaped glass tube and concentrated using a heated vacufuge. Samples from
different part of the ultracentrifuge tube were also collected and similarly
concentrated. Concentrated samples were dissolved in a 4 X SDS solution and
17
separated on a SDS-PAGE protein gel. The protein gel was composed of a 4%
stacking gel and a 10% resolving gel. Stacking gel contained, 1.3 ml of 30%
Acrylamide/Bis, 6.1ml of DDI H2O, 2.5 ml gel buffer and 0.1 ml of 10% w/v SDS.
Resolving gel contained 3.3 ml of 30% Acrylamide/Bis, 4.1ml of DDI H2O, 2.5
ml gel buffer and 0.1 ml of 10% w/v SDS (Mini- PROTEAN/ Cat# 165-8000).
Proteins were visualized using SYPRO RUBY protein gel stain (LONZA/Cat#
50562).
Selecting for a Neutral Lipid Enriched Culture Using Differential Density
Centrifugation
A neutral lipid-enriched culture was prepared by repeating 8 rounds of
enrichment on a wild type culture grown to late log phase. In each round, 10 ml
of culture was centrifuge for 10 minutes at 8397x g using a SORVALL RC-5
ultra-centrifuge and a FIBERLITE F-13-14X50cy rotor, 7 ml of the supernatant
was removed and remaining media/pellet was sonicated for 12 seconds at
amplitude of 13 using a UL TRASONIC PROCESSOR- GEX600. Two ml of the
sonicated culture was over-layed onto 6.3 ml of sterile 65% PERCOLL and
centrifuged for 40 minutes at 26,000x g using an acceleration of 8, deceleration of
2, at a temperature of 220C using a SORVALL Discovery 90 centrifuge and a
SORVALL T-865.1 rotor. Two green bands would be formed and everything
above the bottom band was removed and used to inoculate in A&A/4+Pi
supplemented with MOPS and ammonia. This procedure was performed for eight
rounds after the previous generation had grown to late log phase of growth.
18
Neutral Lipid Formation under Various Growth Conditions
Wild- Type cells were grown in MOPS only (without any nitrogen or
Carbon), MOPS/Ammonia, MOPS/Nitrate, and MOPS/Nitrate/Fructose. Cells
were harvested at log phase (3.8-5.1 g Chla/ml) and stationary phase (31-50 g
Chla/ml) stained with Bodipy and visualized using confocal microscopy (max
projection) followed by Image J analysis (www.http://imagej.nih.gov/).
Experiment was done in triplicates and Chla/ml concentration was normalize for
every stained sample so each would have the same chlorophyll a concentration in
relation to the amount of Bodipy dye (10µl of 0.5µM BODIPY was added to
0.5µg Chla/100µl of cells).
Neutral Lipid Identification
Lipids were extracted from N. punctiforme using two methods in an attempt
to identify the best yield. Total lipids were isolated according to the Bligh and
Dyer method (Bligh, 1959) and only the neutral lipids were isolated according to
Hutchins et al (Hutchins, 2008). The Hutchins’ protocol was slightly modified to
fit the experiment as follows: A pellet containing 180 g Chla (0.1ml) of N.
punctiforme was washed once with diH2O, the pellet was suspended in 0.5ml of
diH2O, and lysed by 4 cycles of sonication on ice for 30 seconds at an amplitude
of 13. To the lysate, 0.5ml of 75:25 v/v of iso-octane/ethyl acetate was added,
vortexed violently for 1 minute, and centrifuged for 7 minutes at 1000 x g. The
top organic layer was removed to a clean glass vial. Another, 0.5ml of 75:25 v/v
of iso-octane/ethyl acetate was added, vortexed violently for 1 minute, and
19
centrifuged for 7 minutes at 1000 x g to the remaining N. punctiforme sample to
make sure all the neutral lipids are extracted. And then the organic layer was dried
under a stream of nitrogen gas.
The dried samples from both the total and neutral lipid extractions were
dissolved in 25μl of hexane and spotted on a silica gel TLC plate (THOMAS
SCIENTIFIC/ Cat#2737B25) that was prewashed (ran) with chloroform and
dried. The spotted samples were separated using a mobile phase consisted of
Toluene / Chloroform / Methanol (85/15/5, V/V/V).. TLC plates were dried after
the mobile phase traveled a desired distance. Dried plates were stained with
Iodine vapors and neutral lipids were identified by comparing the unknown spots
to the standards. (Vegetable oil was used as TAG standard and DAG standards
were purchased from Avanti Polar Lipids, inc #216699) DAG and TAG were
separated on silica gel TLC plates using two different solvent systems that
allowed their migration apart from other confounding impurities in the samples.
TAG separation used Hexane/Ether/Acetic acid (70/30/1, V/V) and DAG
separation used Toluene/Chloroform/Methanol (85/15/5, V/V).
Determination of Fatty Acid Composition
Neutral Lipids were extracted from samples that contained 940 g Chla,
according to Hutchins’ protocol. Cells were sonicated for 4 x 30 seconds but used
10 times as much solvent and cell suspension as the original protocol to
compensate for the extra cells. Dried neutral lipids were turned into fatty acid
methyl esters (FAME) using the following protocol conducted in 8ml glass vials
with Teflon caps. Saponification was accomplished by adding 1 ml of 1N KOH
20
in anhydrous methanol to the dry pellet, heating at 80oC for 30 minutes, then
cooling to room temperature. Methyl esterization was accomplished by adding 1
ml of BCL3-Methanol (12% W/W) and heating 100oC for 10 minutes. After
cooling, 1 ml water and 1 ml hexane was added and vortexed violently for 1
minute to get esters in the nonpolar solvent. The upper organic layer was dried
using a stream of N2 gas. Few crystals of anhydrous sodium sulfate were added to
the drying sample to absorb any water molecules. FAMEs were re-dissolved in 50
µl of hexane and analyzed using gas chromatography- mass spectrometry
Gas chromatography- Mass spectrometry analysis
Wild type cells were grown in triplicates with MOPS/Ammonia, MOPS/Nitrate
and MOPS/Nitrate/Fructose to exponential phase. Neutral lipids of theses cultures
were extracted and turned into FAMEs and analyzed using mass spectrometry
(SHIMADZU/GCMS-QP2010S) with FAME standards (RESTEK/ Cat#35066).
The GC was equipped with a SHRX1-5MS column (0.25mm id x 30m. 0.25 µm
film thickness). The oven temperature was held at 1800C for 1 minute, increased
to 3000C at 120C min-1, with a 3000C final temperature maintained for two
minutes. Helium was used as the carrier gas and 1l of sample was injected in
split mode (1:50). MS detector voltage was set at 0.25 kV, and the samples were
identified using NIST11 and NIST11s libraries.
21
Metabolic Engineering
Mutation of Glycogen Biosynthesis
Primers were designed to delete Npun_R6087 (glucose-1-phosphate
adenylyltransferase) which activates ADP-glucose for polymerization to make
glycogen, by amplify the upstream and the downstream genes of Npun_R6087.
Primers 1 and 2 were used to amplify the upstream region of Npun_R6087 (PCR
1) and primer pair 3 and 4 was used to amplify the downstream stream region of
Npun_R6087 (PCR 2). Primer 3 had complimentary base pairs to primer 2, so the
upstream and the downstream genes of Npun_R6087 could be reattached by PCR,
deleting most of the internal part of Npun_R6087 (PCR 3A). Another primer pair
(5 and 6) was made with restriction enzyme sites SacI and SpeI (PCR 3B) that
would allow cloning this DNA fragment in to a PRL278 plasmid (sacB, Km and
Nm resistant). Reactions and PCR conditions leading to this are listed below
((Primers are identified in the appendix)
PCR reaction 1 (primers 1 & 2) – Amplifying the upstream gene
Per reaction
dd H2O35.8 µl
5X Herculase rxn buffer10 µl
10mM dNTP1.25 µl
Genomic DNA (100ng/1 µl) 1
µl
Primer 1 (10 µm) &
Primer 2 (10 µm) mix
1.25 µl
Herculase polymarase0.7 µl
50 µl
PCR conditions
95 oC
2 minutes
1 cycle
95 oC
40 seconds
57 oC
40 seconds
30 cycles
72 oC
2 minutes
72 oC
3 minutes1 cycle
22
PCR bands were visualized using 1% agarose gel. Resulting PCR products
were cleaned using QIAquickR PCR Purification kit (Qiagen, MD). DNA
concentration was assayed using a Nano-drop ND-1000 and V3.3.0 program.
PCR reaction 2 (Primers 3 & 4) – Amplifying the downstream gene
Per reaction
dd H2O35.8 µl
5X Herculase rxn buffer10 µl
10mM dNTP1.25 µl
Genomic DNA (100ng/1 µl)- 1
µl
Primer 3 (10 µm) &
Primer 4 (10 µm)
1.25 µl
Herculase polymarase0.7 µl
50 µl
PCR conditions
95 oC
95 oC
57 oC
72 oC
72 oC
2 minutes40 seconds
40 seconds
2 minutes
3 minutes-
1 cycle
30 cycles
1 cycle
Successful PCR amplification of the anticipated size was confirmed using
8µl of the PCR reaction in 1% agarose gel electrophoresis. The remaining PCR
products were cleaned using QIAquickR PCR Purification kit (Qiagen, MD). DNA
concentration was assayed using a Nano-drop ND-1000 and V3.3.0 program.
PCR reaction 3A
dd H2O5X Herculase rxn buffer10mM dNTPPCR 1 (70 ng)PCR 2 (70 ng)Herculase polymarase-
(Per reaction)
35.05 µl
10
µl
1.25 µl
1.5 µl
1.5 µl
0.7 µl
50
µl
PCR conditions
95 oC
2 minutes95 oC
30 seconds
44 oC
30 seconds
72 oC
50 seconds
72 oC
3 minutes-
1 cycle
15 cycles
1 cycle
23
PCR reaction 3B
Products of PCR reaction 3A were used to conduct PCR reaction 3B with nested
primers (Primers 5 & 6). Amounts listed are per reaction (3 different reactions
were performed for 3B, then combined for cleaning prior to digestion)
dd H2O5X Herculase rxn buffer10mM dNTPPCR 3APrimer 5 (10 µm) &
Primer 6 (10 µm)
Herculase polymarasePCR conditions
95 oC
95 oC
57 oC
72 oC
72 oC
24.8 µl
7
µl
1.25 µl
15 µl
1.25 µl
0.7 µl
50 µl
2 minutes30 seconds
30 seconds
1.5 minutes
3 minutes-
1 cycle
30 cycles
1 cycle
Successful PCR amplification of the anticipated size were confirmed using 8µl of
the PCR reaction in 1% agarose gel electrophoresis. The remaining PCR products
were cleaned using QIAquickR PCR Purification kit (Qiagen, MD). DNA
concentration was assayed using a Nano-drop ND-1000 and V3.3.0 program.
Restriction enzyme digestion reaction
Products of PCR reaction 3B and the purified plasmid (pRL278) was digested as
below (volumes listed are in µl)
24
Restriction enzyme digestion reaction
Products of PCR reaction 3B and the purified plasmid (pRL278) was digested as
below (in µl)
Water
10X NEB
buffer #1
10X
BSA
pRL278
(325ng)
Insert- 3B
(19ng)
Enzyme
Total
SacI
14.5
Plasmid
SpeI
13.5
SacI/SpeI
12.5
sacI/SpeI
24
Insert
SacI/SpeI
13
2
2
2
4
5
2
2
2
4
5
1.5
1.5
1.5
6
0
0
0
0
1
0
2
0
2
25
2
20 µl
20 µl
20 µl
40 µl
50 µl
The reactions were incubated for 45 minutes at 37oC and the success of the
digestion was confirmed by gel electrophoresis (1% Agarose) of the 20 µl
reactions. Digested products were cleaned using QIAquickR PCR Purification kit
(Qiagen, MD). DNA concentration was assayed using a Nano-drop ND-1000 and
V3.3.0 program.
Digested and cleaned fragments were ligated as below (in µl)
Water
5X ligase buffer
Plasmid(22ng/µl)
Insert (8.6ng/µl)
T4 DNA ligase
Experiment
1
5
3
10
1
20 µl
Control
11
5
3
0
1
20 µl
The reaction were kept at room temperature for 60 minutes and transferred to the
door of the 4oC refrigerator until transformation.
Ligated reactions were transformed into 200 µl CaCl2 competent E. coli
cells (DH5 alpha MCR) using 4 µl DNA (Ligation mix and the control to a
separate reaction), gently mixing and setting on ice for 20 minutes, then heat
25
shocking the cells at 42-43oC for 90 seconds and return to ice for 1-5 minutes to
recover. After recovery, 1ml SOC was added and the cells allowed to recover for
1 hour at 37oC in the micro centrifuge tube in the rolling incubator. Transformed
cells were plated on selective LB plates containing Kanamycin 50 mg/ml.
Colony PCR to confirm inserts
29 colonies were randomly selected on overnight grown (37oC), LB/Km plates.
Colony PCR was performed on these colonies.
PCR conditions per reaction
dd H2O10X PCR PCR buffer10mM dNTPs
25mM MgCl2Primer pRL1&
Primer pRL2 (each at10 µm)
Taq polymarase-
36 µl
5 µl
1 µl
3 µl
2 µl
3 µl
50 µl
95 oC
5 minutes1 cycle
o
95 C
1 minute
57 oC
1 minute
30 cycles
o
72 C
3 minutes
72 oC
3 minutes1 cycle
PCR bands were visualized using 1% agarose gel.
Conjugation
Conjugation was done using three randomly selected positive clones according to
standard procedures (Summers,1996). A true mutant was discovered after
selecting for the primary and secondary N. punctiforme recombinant mutants
(Summers, 1996).
26
PCR conditions to identify the true mutant
dd H2O10X PCR rxn bufferDMSO
1% Tween
BSA(100ug/ml)
10mM dNTP25mM MgCl2Primer P5 (10 µm) &
Primer P6 (10 µm)
Taq polymarase-
25 µl
5 µl
1 µl
5 µl
5 µl
1 µl
3 µl
2 µl
3 µl
50 µl
95 oC
2 minutes1 cycle
95 oC
1 minute
57 oC
1 minute
35 cycles
68 oC
4 minutes
68 oC
10 minutes1 cycle
PCR bands were visualized following electrophoresis using a 1% agarose gel.
Mutation of Cyanophycin Synthesis
Primers were designed to delete NpR5823 by amplify the upstream and the
downstream region of NpR5823 (Primers are identified in the appendix). Primers
1 and 2 were used to amplify the upstream gene of NpR5823 (PCR 1) and primer
pair 3 and 4 was used to amplify the downstream stream gene of NpR5823 (PCR
2). Primer 3 had complimentary base pairs to primer 2, so the upstream and the
downstream genes of NpR5823 could be reattached by PCR through
complementary priming, deleting the internal section of NpR5823 (PCR 3A).
Another primer pair 5 and 6 was made with restriction enzyme sites BamH1 and
Xho1 (PCR 3B) that would allow cloning this DNA fragment in to a PRL278
plasmid (sacB, Km and Nm res).
27
PCR reaction 1 (primers 1 & 2) – Amplifying the upstream region (per reaction)
dd H2O35.8 µl
5X Herculase rxn buffer10 µl
10mM dNTP1.25 µl
Genomic DNA (100ng/1 µl)- 1
µl
Primer 1 (10 µm) &
Primer 2 (10 µm)
1.25 µl
Herculase polymarase0.7 µl
50 µl
PCR conditions
95 oC
2 minutes95 oC
40 seconds
54 oC
40 seconds
72 oC
2.5 minutes
72 oC
3 minutes-
1 cycle
30 cycles
1 cycle
PCR bands were visualized using 1% agarose gel. Resulting PCR products were
cleaned using QlAquickR PCR Purification kit (Qiagen, MD). DNA concentration
was assayed using a Nano-drop ND-1000 and V3.3.0 program.
PCR reaction 2 (Primers 3 & 4) – Amplifying the downstream gene (per reaction)
dd H2O35.8 µl
5X Herculase rxn buffer10 µl
10mM dNTP1.25 µl
Genomic DNA (100ng/1 µl)- 1
µl
Primer 3 (10 µm) &
Primer 4 (10 µm)
1.25 µl
Herculase polymarase0.7 µl
50 µl
PCR conditions
95 oC
2 minutes1 cycle
95 oC
40 seconds
54 oC
40 seconds
30 cycles
72 oC
1.5 minutes
72 oC
3 minutes1 cycle
PCR bands were visualized using 1% agarose gel. Resulting PCR products were
cleaned using QlAquickR PCR Purification kit (Qiagen, MD). DNA concentration
was assayed using a Nano-drop ND-1000 and V3.3.0 program.
28
PCR reaction 3A
PCR reaction 1 and 2 were combined and amplified.
Per reaction
dd H2O35.05 µl
5X Herculase rxn buffer10
µl
10mM dNTP1.25 µl
PCR 1 (70ng/µl)
1.5 µl
PCR 2 (70ng/µl)
1.5 µl
Herculase polymarase0.7 µl
50
µl
3 different reactions were performed for PCR reaction 3B.
PCR conditions
95 oC
2 minuteso
95 C
30 seconds
44 oC
30 seconds
72 oC
50 seconds
o
72 C
3 minutes-
1 cycle
15 cycles
1 cycle
PCR reaction 3B
Products of PCR reaction 3A were used to conduct PCR reaction 3B with nested
primers (Primers 5 & 6).
Per reaction
dd H2O5X Herculase rxn buffer10mM dNTPPCR 3APrimer 5 (10 µm) &
Primer 6 (10 µm)
Herculase polymarase-
24.8 µl
7
µl
1.25 µl
15 µl
1.25 µl
0.7 µl
50 µl
3 different reactions were performed for PCR reaction 3B, and then combined to
have enough material for the following steps.
PCR conditions
95 oC
2 minuteso
95 C
30 seconds
54 oC
30 seconds
o
72 C
3 minutes
72 oC
3 minutes-
1 cycle
30 cycles
1 cycle
29
PCR bands were visualized using 1% agarose gel. Resulting PCR products were
cleaned using QIAquickR PCR Purification kit (Qiagen, MD). DNA concentration
was assayed using a Nano-drop ND-1000 and V3.3.0 program.
Restriction enzyme digestion reaction
Products of PCR reaction 3B and the purified plasmid (pRL 278) was digested as
below (in µl)
Plasmid
BamH1
Water
10X Fast Digest
buffer
pRL278 (50ng)
Insert3B(200ng)
Fast Alc. Pho.
Enzyme
Total
Xho1
BamH1/Xho1
None
32.4
Insert
BamH1/
Xho1
27.2
31.4
31.4
27.7
4
4
4
2.6
2.6
10.3(200ng)
4
2.6
0
0
0
0
6.8
1
1
1
1
40
1
2
40
1
0
0
2
40 µl
40 µl
40 µl
4
0
The reactions were incubated for 45 minutes at 37oC and the success of the
digestion was confirmed by gel electrophoresis of the 20µl reactions on a 1%
agarose gel. Digested products were cleaned using QIAquickR PCR Purification
kit (Qiagen, MD). DNA concentration was assayed using a Nano-drop ND-1000
and V3.3.0 program.
Digested fragments were ligated as below using Fermentas Rapid Ligation Kit (in
µl)
Water
5X ligase buffer
Plasmid(100 ng)
Insert (100 ng)
T4 DNA ligase
Experiment
11.3
4
2.2
1.5
1
20 µl
Control
12.8
4
2.2
0
1
20 µl
The reactions were kept at room temperature for 30 minutes.
30
Ligation reactions were transformed in to CaCl2 competent E. coli cells (DH5
alpha MCR) as described above.
Eight colonies were randomly selected following overnight grown (37oC)
on LB/Km plates, the colonies inoculated to LB broth under Km selection, the
plasmids isolated using a Promega SV Miniprep kit and ~350 ng digested with
BamHI/XhoI to test for the plasmid with the proper insert.
Conjugation
Conjugal transfer from E. coli to N. punctiforme was performed using three
randomly selected colonies by Angelica Cardenas according to a standard
protocol (Summers, 1996).
31
Results
Proteins involved in neutral lipid droplet formation
The mechanism of neutral lipid formation in N. punctiforme is unknown.
To identify proteins involved in this process, neutral lipids of N. punctiforme were
enriched using centrifugation and 0.5M sucrose solution with an over lay without
sucrose. After ultra centrifugation a yellowish hydrophobic layer was observed in
the over- lay (Figure 5). The proteins associated with the lipid enriched fraction
and the other fractions were separated using a 10% SDS-PAGE gel. It was
hypothesized that the upper fraction of the tube should be enriched for proteins
associated with neutral and other lipids due to the low buoyancy of the lipids,
whereas the lower parts should have fewer. Analysis of the protein gel separating
the various fractions however did not reveal protein bands unique to the upper
layer.
Crude Ladder
Figure 5- Separation of proteins on a protein gel (Total and lipid enriched
proteins)
32
Selecting for a Neutral Lipid Enriched Culture Using Ultra Centrifugation
A culture that overproduces neutral lipids was developed through Percoll
selection, using the working hypothesis that the presence of more lipids would be
correlated with more buoyancy. As illustrated in Figure 6, lightly sonicated wild
type cells form two bands after ultra centrifugation. The top light green band
contains mostly dead cell material whereas the lower thick green band contains
most of the cells. Cells between these two bands were collected hoping that those
cells would have the most neutral lipid content. The selected fraction was
inoculated into fresh media, grown to late log phase and another round of Percoll
selection was performed. After eight rounds of Percoll selection, neutral lipids
were stained with Bodipy and visualized by laser confocal microscopy.
Figure 6- Separating the cells with highest lipid content
Neutral Lipid staining and Visualization
Neutral lipids of N. punctiforme were stain with Bodipy and visualized by
Confocal laser microscopy. Bodipy goes into neutral lipids within seconds and
fluoresce as shown in Figure 7. Lipid area per cell was quantified using ImageJ
from the maximum projected Z-stack images. Using the free hand selection on
Image J a line was traced through the edges of a filament (Figure 8) and the pixels
within that area was calculated. The same was done on all the lipid droplets in the
33
filament being analyzed (illustrated for the lipid globule denoted by the arrow in
Figure 9).
Figure 7- Stained with Bodipy and visualized using maximum projection on
confocal microscope.
Quantification
Figure 8- Measuring the area of the filaments using Image J
Figure 9- Measuring the area of the lipid globules using Image J
Selecting for a Neutral Lipid Enriched Culture Using Ultra Centrifugation
After eight rounds of Percoll selection a culture containing filaments with
a higher concentration of neutral lipids were observed (Figure 10). This culture
was named as the selection 8. Quantification of neutral lipids of randomly
selected filaments in both wild type and selection 8 grown similarly suggested
that the selection 8 contains twice as much neutral lipids (Figure 11).
34
Wild Type
Selection 8
Figure 10- Neutral lipid staining with the fluorescent dye Bodipy and
visualization using laser confocal microscopy
Neutral Lipid Content of Selection 8 Vs Wild-Type Cells
Neutral Lipid Area/Cell Area (%)
14.00
12.00
10.84
10.00
8.00
6.00
4.87
4.00
2.00
0.00
Selection 8 (late stationary phase)
Wild-Type (late stationary phase)
Cell Type
Figure 11- Quantification of stained area for 50 cells (grown with
MOPS/ammonia/nitrate to stationary phase) with ImageJ.
35
Neutral Lipid Formation in Relative to the Nitrogen and Carbon Utilization
Wild type cells were grown with different nitrogen and carbon sources to study
the effect of growth conditions on neutral lipid formation (Figure 12). According
to the results, the highest increase in neutral lipids was observed in cultures grown
under nitrogen-fixing conditions going from log phase to stationary phase (3.3fold increase). Cultures grown in MOPS/Ammonia increased 2.4-fold during the
transition from exponential to stationary phase of growth. The MOPS/Nitrate
culture increased only 1.8- fold, and the MOPS/Nitrate/Fructose grown cultures
actually decreased the area of lipids per cell upon entering the stationary phase of
growth.
The highest neutral lipid production in log phase cultures was produced by
MOPS/Nitrate/Fructose grown cultures. The exogenous sugars increased the
production of neutral lipids in log phase but not in stationary phase. The highest
yield of neutral lipids in stationary phase were produced by both MOPS/Nitrate
and MOPS/Nitrate/Fructose grown cultures. It should be noted that the reduction
in overall production of neutral lipids in relation to the cell size in
MOPS/Nitrate/Fructose cultures is misleading, and is actually due to the increased
size of cell grown in fructose (data not shown).
36
(Area of the Lipid Droplets/ Area of the
Cells)*100
Neutral Lipid formation Under Different Nitrogen and Carbon Sources
4.5
3.90
4
3.27
3.5
3.13
3
3.06
2.73
2.5
2.17
2
1.5
1.39
0.92
1
0.5
0
MA/Log
MA/Sta
MN/Log
MN/Sta1 MNF/Log
MNF/Sta
MOPS/Log
MOPS/Sta
Growth Media/Growth Stage
Figure 12- Relative neutral lipid area relative to the cell size (N>100) was
determined from Bodipy stained confocal Z-stack projection images measured by
Image J. Cultures were grown in MOPS/Ammonia (MA), MOPS/Nitrate (MN),
MOPS/Nitrate/Fructose (MNF) or MOPS only. Cells were measured during log
phase (3.8-5.1 g Chla/ml) and during stationary phase (31-50 g Chla/ml)
Identifying the Neutral Lipids Formed in Nostoc punctiforme
Lipids of N. punctiforme were extracted according to both the Bligh & Dyer and
Hutchins protocols, and separated on TLC plates using appropriate solvent
systems. Hutchins’ protocol had a better separation of neutral lipids compared
with Bligh & Dyer since Hutchins’ method only extracts the neutral lipids
compared to total lipids from Bligh & Dyer (Figure 13). Upon a closer look at the
red ovals in Figure 13, shows a brighter spot for DAG extracted according to
Hutchins’ protocol. According to the TLC results, both triacylglycerol and
diacylglycerol exist in Nostoc punctiforme (Figure 14 and 15).
Hexane/Ether/Acetic acid (70/30/1, V/V) and Toluene/Chloroform/ Methanol
37
(85/15/5, V/V) produced the best separation of TAG and DAG from N.
punctiforme neutral lipid extractions.
Both DAG and TAG could not be separated on the same TLC plate using
this solvent system due to either the chlorophyll contamination of DAG (Figure
14) or TAG running with the solvent front (Figure 15). To enable resolution of
each neutral lipid type on the same TLC plate, different mobile phases were tested
to separate TAG and DAG
1
1
2
3
4
5
6
Figure 13- BD= Bligh and Dyer method (50 µl of the sample was spotted), NL=
Neutral lipid extraction method (50 µl of the sample was spotted), Vegetable oil
38
was used as Triacylglycerol (TAG) standard (50 µg of the standard was spotted),
Diacylglycerol (DAG) standard was obtained through Avanti Polar Lipids, Inc.
(Cat# 800815C) and 50 µg of the standard was spotted on the TLC plate. Silica
coated TLC plates were obtained from SIGMA – ALDRICH (Cat#2737B25).
Toluene/Chloroform/Methanol (85/15/5, V/V) was used as the mobile phase.
Separating DAG and TAG Using Proper Solvents
The Hexane/Ether/Acetic acid (70/30/1, V/V/V) solvent system successfully
moved the TAG away from the migration front, however the DAG still migrated
close to another contaminant (assumed to be chlorophyll due to the color green
and neutral lipid properties of phytol groups in chlorophyll) extracted by the
neutral lipid protocol (Figure 14). The Toluene/Chloroform/Methanol (85/15/5,
V/V) solvent system successfully separated DAG away from the chlorophyll;
however the TAG still migrated too close to the migration front to allow adequate
resolution from other unknown contaminants (Figure 15). In both Figure 14 and
15 neutral lipids were isolated from biological samples. Spots being the same
intensity for all three samples suggest that the extractions are reproducible.
39
TAG- Hexane/Ether/Acetic acid (70/30/1, V/V)
TAG
50µg
DAG
50µg
WT
WT
WT
Figure 14- Neutral Lipids in MOPS/Ammonia grown wild-type cells were
extracted according to the neutral lipid extraction protocol.
40
DAG- Toluene/Chloroform/Methanol (85/15/5, V/V)
TAG
50µg
DAG
50µg
WT
WT
WT
Figure 15. Neutral Lipids in MOPS/Ammonia grown wild-type cells were
extracted according to the neutral lipid extraction protocol.
Fatty Acid Composition of the Neutral Lipids
The wild type cells were grown in MOPS/Ammonia, MOPS/Nitrate, and
MOPS/Nitrate/Fructose to study the effect of growth condition on carbon chains
of neutral lipids. To determine the fatty acid composition of extracted neutral
-
lipids, samples were converted to FAMEs and subjected to gas chromatographymass spectrometry (GC-MS). FAMEs were extracted with ought being
contaminated by cholorophyll (Figure 16). The GC-MS results indicate that N.
punctiforme neutral lipids consist of five major hydrocarbon chains: C16
41
[Hexadecenoic acid (Palmitic acid)], C16:1 (9-Hexadecenoic acid), C18:0
[Octadecanoic acid- (Stearic acid)], C18:2 [9,12- Octadecadienoic acid
(Lenolelaidic acid)] and C18:3 [9,12,15- Octadecatrienoic acid (Linolenic acid)]
(Figures 17-31).
In general, FAME samples from N. punctiforme eluted from the GC
column identically to the known standards, and correctly identified by the
software library (NIST11 and NIST11s). There appears to be confusion with the
C18:3 ion chromatographs (Figures 20, 25 and 30), however it can be explained
by a closer look at the data. Based on the chromatograph for the standard there
seem to be no peak where C18:3 suppose to be. But at a closer look there is a
small shoulder at the bottom of the C18:1 (which is not present in N. punctiforme
samples) that represents C18:3 (Figures 20, 25 and 30). This is in agreement with
the composition of the standard which is 14% C18:1 and only 2% C18:3. C18:3
shows up too close to C18:1 in the SHRX1-5MS column and the low
concentration of C18:3 in the standard compared to C18:1 hinders it from being
identified correctly. In the N. punctiforme samples, there is little C18:1 present,
thus allowing the C18:3 peak to be observed at the correct elution time, and
allows for its correct identification by the software (Figures 20, 25 and 30).
The GC data from the GC-MS analysis of all samples was extracted in an
attempt to quantify changes in neutral lipid fatty acid composition changed with
growth condition. Peak areas for biological triplicate samples which represents
the amount of each FAME were averaged (Table 1) and plotted as a percent of
total neutral lipid in each sample (Figure 32 ). According to the results, carbon
42
chains of neutral lipids in wild type cells could be altered by changes in growth
conditions. C16 had the highest concentration regards to any condition. Cells
grown in MN had the highest concentration of saturated C16 and C18’s. Cells
grown in MA had the highest C16:1 and the lowest C18 concentration while cells
grown in MNF had the highest concentration of C18:2 and C18:3 (Figure 33).
Neutral Lipids
Figure 16- Fatty acid methyl esters of neutral lipids.
43
Gas Chromatography- Mass Spectrometry
Figure 17- GC-MS results of neutral lipids extracted from N. punctiforme wild
type cells grown in MOPS/Ammonia (MA) from the exponential growth phase.
The top graph shows the total gas chromatograms for Mops/Ammonia triplicates
over laid on the standard. The second graph shows the selection of C16:0 and
C16:1 region of the gas chromatograph with a vertical line denoting the C16:1
peak. The bottom figures show m/z values for the C16:1 standard and sample
triplicate.
44
Figure 18- GC-MS results of neutral lipids extracted from N. punctiforme wild
type cells grown in MOPS/Ammonia (MA) from the exponential growth phase.
The top graph shows the total gas chromatograms for Mops/Ammonia triplicates
over laid on the standard. The second graph shows the selection of C16:0 and
C16:1 region of the gas chromatograph with a vertical line denoting the C16
peak. The bottom figures show m/z values for the C16 standard and sample
triplicate.
45
Figure 19 - GC-MS results of neutral lipids extracted from N. punctiforme wild
type cells grown in MOPS/Ammonia (MA) from the exponential growth phase.
The top graph shows the total gas chromatograms for Mops/Ammonia triplicates
over laid on the standard. The second graph shows the selection of C18:2, C18:3
and C18 region of the gas chromatograph with a vertical line denoting the C18:2
peak. The bottom figures show m/z values for the C18:2 standard and sample
triplicate.
46
Figure 20 - GC-MS results of neutral lipids extracted from N. punctiforme wild
type cells grown in MOPS/Ammonia (MA) from the exponential growth phase.
The top graph shows the total gas chromatograms for Mops/Ammonia triplicates
over laid on the standard. The second graph shows the selection of C18:2, C18:3
and C18 region of the gas chromatograph with a vertical line denoting the C18:3
peak. The bottom figures show m/z values for the C18:3 standard and sample
triplicate.
47
Figure 21- GC-MS results of neutral lipids extracted from N. punctiforme wild
type cells grown in MOPS/Ammonia (MA) from the exponential growth phase.
The top graph shows the total gas chromatograms for Mops/Ammonia triplicates
over laid on the standard. The second graph shows the selection of C18:2, C18:3
and C18 region of the gas chromatograph with a vertical line denoting the C18
peak. The bottom figures show m/z values for the C18 standard and sample
triplicate.
.
48
Figure 22- GC-MS results of neutral lipids extracted from N. punctiforme wild
type cells grown in MOPS/Nitrate (MN) from the exponential growth phase. The
top graph shows the total gas chromatograms for MOPS/Nitrate triplicates over
laid on the standard. The second graph shows the selection of C16:0 and C16:1
region of the gas chromatograph with a vertical line denoting the C16:1
peak. The bottom figures show m/z values for the C16:1 standard and sample
triplicate.
49
Figure 23- GC-MS results of neutral lipids extracted from N. punctiforme wild
type cells grown in MOPS/Nitrate (MN) from the exponential growth phase. The
top graph shows the total gas chromatograms for MOPS/Nitrate triplicates over
laid on the standard. The second graph shows the selection of C16:0 and C16:1
region of the gas chromatograph with a vertical line denoting the C16 peak. The
bottom figures show m/z values for the C16 standard and sample triplicate.
50
Figure 24- GC-MS results of neutral lipids extracted from N. punctiforme wild
type cells grown in MOPS/Nitrate (MN) from the exponential growth phase. The
top graph shows the total gas chromatograms for MOPS/Nitrate triplicates over
laid on the standard. The second graph shows the selection of C18:2, C18:3 and
C18 region of the gas chromatograph with a vertical line denoting the C18:2
peak. The bottom figures show m/z values for the C18:2 standard and sample
triplicate.
51
Figure 25- GC-MS results of neutral lipids extracted from N. punctiforme wild
type cells grown in MOPS/Nitrate (MN) from the exponential growth phase. The
top graph shows the total gas chromatograms for MOPS/Nitrate triplicates over
laid on the standard. The second graph shows the selection of C18:2, C18:3 and
C18 region of the gas chromatograph with a vertical line denoting the C18:3
peak. The bottom figures show m/z values for the C18:3 standard and sample
triplicate.
52
Figure 26- GC-MS results of neutral lipids extracted from N. punctiforme wild
type cells grown in MOPS/Nitrate (MN) from the exponential growth phase. The
top graph shows the total gas chromatograms for MOPS/Nitrate triplicates over
laid on the standard. The second graph shows the selection of C18:2, C18:3 and
C18 region of the gas chromatograph with a vertical line denoting the C18
peak. The bottom figures show m/z values for the C18 standard and sample
triplicate.
53
Figure 27- GC-MS results of neutral lipids extracted from N. punctiforme wild
type cells grown in MOPS/Nitrate/Fructose (MNF) from the exponential growth
phase. The top graph shows the total gas chromatograms for
MOPS/Nitrate/Fructose triplicates over laid on the standard. The second graph
shows the selection of C16:0 and C16:1 region of the gas chromatograph with a
vertical line denoting the C16:1 peak. The bottom figures show m/z values for the
C16:1 standard and sample triplicate.
54
Figure 28- GC-MS results of neutral lipids extracted from N. punctiforme wild
type cells grown in MOPS/Nitrate/Fructose (MNF) from the exponential growth
phase. The top graph shows the total gas chromatograms for
MOPS/Nitrate/Fructose triplicates over laid on the standard. The second graph
shows the selection of C16:0 and C16:1 region of the gas chromatograph with a
vertical line denoting the C16 peak. The bottom figures show m/z values for the
C16 standard and sample triplicate.
55
Figure 29- GC-MS results of neutral lipids extracted from N. punctiforme wild
type cells grown in MOPS/Nitrate/Fructose (MNF) from the exponential growth
phase. The top graph shows the total gas chromatograms for
MOPS/Nitrate/Fructose triplicates over laid on the standard. The second graph
shows the selection of C18:2, C18:3 and C18 region of the gas chromatograph
with a vertical line denoting the C18:2 peak. The bottom figures show m/z values
for the C18:2 standard and sample triplicate.
56
Figure 30- GC-MS results of neutral lipids extracted from N. punctiforme wild
type cells grown in MOPS/Nitrate/Fructose (MNF) from the exponential growth
phase. The top graph shows the total gas chromatograms for
MOPS/Nitrate/Fructose triplicates over laid on the standard. The second graph
shows the selection of C18:2, C18:3 and C18 region of the gas chromatograph
with a vertical line denoting the C18:3 peak. The bottom figures show m/z values
for the C18:3 standard and sample triplicate.
57
Figure 31- GC-MS results of neutral lipids extracted from N. punctiforme wild
type cells grown in MOPS/Nitrate/Fructose (MNF) from the exponential growth
phase. The top graph shows the total gas chromatograms for
MOPS/Nitrate/Fructose triplicates over laid on the standard. The second graph
shows the selection of C18:2, C18:3 and C18 region of the gas chromatograph
with a vertical line denoting the C18 peak. The bottom figures show m/z values
for the C18 standard and sample triplicate.
58
Media
MOPS/Ammonia
MOPS/Nitrate
MOPS/Nitrate/Fructose
Carbon
length
C16:1
C16
C18:2
C18:3
C18
C16:1
C16
C18:2
C18:3
C18
C16:1
C16
C18:2
C18:3
C18
Sample
1
19.94
39.68
16.55
19.57
4.26
15.93
43.86
14.77
14.37
11.07
16.91
35.62
18.69
20.03
8.75
Sample
2
27.36
38.99
13.34
16.48
3.83
15.44
42.09
16.36
14.53
11.58
18.95
30.71
23.51
21.59
5.24
Sample
3
23.44
40.92
14.87
17.03
3.74
13.86
44.35
14.71
13.15
13.93
18.56
31.58
20.64
22.56
6.66
Average
23.6
39.9
14.9
17.7
3.9
15.1
43.4
15.3
14.0
12.2
18.1
32.6
20.9
21.4
6.9
SD
3.71
0.98
1.61
1.65
0.28
1.08
1.19
0.94
0.75
1.53
1.08
2.62
2.42
1.28
1.77
Stan.
Error
2.14
0.56
0.93
0.95
0.16
0.62
0.69
0.54
0.44
0.88
0.63
1.51
1.40
0.74
1.02
Table 1- Integrated area under the curve for each carbon chain for all the growth
conditions was calculated.
MOPS/Ammonia
MOPS/Nitrate
C18:3
C18:2
C18
C16:1
C16
C18:3
C18:2
C18
C16:1
C16
C18:3
C18:2
C18
C16:1
50.0
45.0
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
C16
% of Total
Fatty Acid Composition of Neutral Lipids
MOPS/Nitrate/Fructose
Fatty Acid Chains per Growth Condition
Figure 32- Concentration of fatty acid chains in wild type cells grown in different
growth conditions.
59
Mutating Glycogen and Cyanophycin Biosynthesis
Although algae have been the organism of choice for biodiesel production
due to their natural high yield of neutral lipids, they are eukaryotic organisms that
are harder to genetically engineer. Cyanobacteria however are prokaryotic
organisms with a facile genetic system. We hypothesize that although
cyanobacteria do not make large quantities of neutral lipids, they could be
genetically modified to increase their production by blocking non-essential carbon
storage pathways. To this end, a glucose-1-phosphate adenylyltransferase
deletion plasmid was created by PCR mediated ligation (Figure 33) and
conjugated into Nostoc punctiforme. Following selection and screening for
double recombinants, a mutant unable to synthesize glycogen was
identified. This can bee seen from a gel showing a colony PCR product from
wild-type and mutant strains of N. punctiforme (Figure 34). The P5 and P6
primers (refer to appendix) produced a 3365 bp PCR fragment in the wild-type
strain, and only a 2324 bp PCR fragment in the mutant strain, indicating a
deletion occurred in the genome between this primer pair.
A second mutation in cyanophycin synthatase (NpR5823) that is
expected to block cyanophycin (a non-ribosomally produced amino acid polymer
used for carbon and nitrogen storage in cyanobacteria) production is underway.
Cyanophycin synthatase was successfully deleted by PCR amplification of
upstream gene (with primer pair 1 and 2) and downstream gene (with primer pair
3 and 4) of NpR5823. PCR 1 (primers 1&2) yield a PCR product of 1310 bp and
PCR 2 (primers 3 & 4) yield a product of 1478 bp (data not shown). Both PCR 1
60
and 2 were combined through PCR reaction 3A deleting the internal section of
NpR5823 (data not shown). Products of PCR 3A was amplified with the primer
pair 5 and 6 that contained restriction enzyme sites BamH1 and Xho1 (PCR 3B)
that would allow cloning this DNA fragment in to a PRL278 plasmid (sacB, Km
and Nm res). PCR 3B produced a DNA fragments that is 1901 bp long (data not
shown). This fragment was cloned in to PRL 278 plasmid. A plasmid digestion
was performed to assure proper ligation of the insert to the plasmid. Figure 35
confirmed the proper insertion of the insert in to the plasmid by releasing the 1901
bp insert when digested. Currently, single recombinants following conjugation of
this plasmid are being selected as well.
1
2
3 4 5 6 7 8
Figure 33- Confirming the glycogen mutant plasmid construct by colony PCR
61
L
M WT
3334 bp- Wild Type
2168 bp- Mutant
Figure 34- Confirming the double recombinant mutant by colony PCR using
primers 5 and 6. PCR products were separated on a 1% agarose gel and
stained with ethidium bromide.
Cyanophycin Mutant
Plasmid +Insert (No
restriction enzymes)
Plasmid + insert-------------------------------
Plasmid- 5941bp
Insert - 1901bp
Figure 35- Confirming the cyanophycin mutant plasmid construct by plasmid
digest
62
Discussion
Proteins Involved in Neutral Lipid Formation
Attempt to isolate proteins associated with neutral lipid droplets did not
yield any unique bands on the neutral lipid droplet enriched fraction (Figure 5).
Efforts to purify the proteins by separating the upper fraction on 150mM KCl did
not yield any bands on a protein gel. Identifying each band on the upper fraction
using LC-MS could be very expensive due to the number of bands, and due to the
fact that they were not unique to this layer. Because this direct route to identifying
proteins associated with lipid globules was unsuccessful, an alternative
physiological/genetic route was taken to identify the genes involved in neutral
lipid formation.
Selecting for a Neutral Lipid Enriched Culture Using Buoyant Density
The alternate route involved finding genes associated with neutral lipids
was to compare a neutral lipid over-producing strain to a wild type strain using
microarray analysis. The working hypothesis was that lipid droplet associated
proteins in over-expression strains would be up-regulated due to transcriptional
increases. Quantification of lipid droplet area per cell from digital images
indicated that the selected culture produced twice as many neutral lipid droplets as
the wild type strain under similar growth conditions (Figure 11). A wild type
culture has filaments with varying concentrations of neutral lipid droplets. By
eight rounds of selection I have enriched for a culture that has a higher
concentration of neutral lipid droplets per cell. However, even in this culture
there are filaments with different neutral lipid content. Currently, single colonies
63
of this mix culture are being selected to find a super-producer. Once this superproducer has been identified, microarray analysis would be done on this strain
with another strain that does not produce as much neutral lipids. Once the neutral
lipid droplet forming pathways are known, the genes that are responsible for
neural lipid droplet formation would be over expressed in N. punctiforme, and
expected to yield a higher amount of lipid droplets per cell.
Neutral Lipid Formation in Relative to the Nitrogen and Carbon Utilization
Neutral lipid droplet formation changes with the growth condition. Most
of the published work on growth conditions and neutral lipid droplet formation in
algae has been done by starving the cell for nutrients. This work suggests that the
neutral lipid droplet concentration of N. punctiforme also changes with the kind of
nutrient that’s being fed (Figure 12).
Identifying the Neutral Lipids Formed in N. punctiforme
According to the TLC results both triacylglycerol and diacylglycerol exist
in Nostoc punctiforme. The three extractions that were done on one biological
sample (Figure 14 and 15). The results showed that the spot size of the TAG and
DAG remains about the same. This suggests that the extraction method is
consistent. A new solvent system that contains Iso-octane/Ethyl acetate (75/25
v/v) has been developed to separate both TAG and DAG on the same plate. This
method also does a better job in preventing chlorophyll from migrating with DAG
and slows migration of TAG away from the migration front (data not shown).
64
Fatty Acid Composition Neutral Lipids
According to GC-MS work cyanobacterial neutral lipids consist of five
major hydrocarbon chains: C16 [Hexadecenoic acid (Palmitic acid)], C16:1 (9Hexadecenoic acid), C18 [Octadecanoic acid- (Stearic acid)], C18:2 [9,12Octadecadienoic acid (Lenolelaidic acid)] and C18:3 [9,12,15- Octadecatrienoic
acid (Linolenic acid)]. The composition of the fatty acid chains of neutral lipids
could be changed with the growth media. When exogenous carbon is
supplemented to cells grown in MN, most of the saturated C18’s turn into
unsaturated C18:2 and C18:3 (Figure 33). The preliminary work done with the
growth conditions suggests that fatty acid desaturases could be controlled with the
growth condition. This experiment was done with a single extraction of biological
triplicates. The consistency of the fatty acid concentration for the FAME species
suggests that the results are reproducible. More work could be done on different
growth conditions to extend these results.
Metabolic Engineering
Cyanobacterial metabolic pathways were altered to favor neutral lipid
production. The mutant that is unable to produce glycogen tended to exhibit short
filaments, were unable to grow under nitrogen-fixing conditions, and fragmented
and died upon reaching stationary phase. Complementation done on this mutant
by Areyh Solomon (Summers’ Lab, CSUN) failed to fix this issue, indicating a
secondary mutation likely occurred to cause this phenotype. Conjugation to
produce this mutant was done again and currently being screened for single
recombinant. Conjugation to produce the cyanophycin synthase mutant has also
65
been done. Once true mutants are obtained, the neutral lipid droplet concentration
of the mutants in log and stationary phase will be compared to the wild type. Also,
since the mutagenesis procedure does not leave behind any antibiotic resistance
marker, each mutant will be mutagenized again to remove the other storage
pathways and similarly analyzed.
66
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69
Appendix
Details of deletion mutagenesis for inactivation of NpF6087 (glucose-1-phosphate
adenylyltransferase) and NpR5823 (cyanophycin synthetase)
Glycogen Mutagenesis
Glycogen mutagenesis primers
AVP6087mutP1
CGAGAAATCCATCTTTGTGG
AVP6087mutP2
ATAGGTACGGGCGATGTGAC
AVP6087mutP3
GTCACATCGCCCGTACCTATAATGCTGTAATTCCCGATGG
AVP6087mutP4
GTAACTTTGTCGCCCACCTC
AVP6087mutP5
GAAGGAGCTCAATTTCTCGCTAATCGCTTCC
AVP6087mutP6
GCAGACTAGTCAAAGCGCCCTAGCAACTC
Strategy for inactivating glycogen synthesis
Npun_R6087 (glgC) encodes glucose-1-phosphate adenylyltransferase
[EC:2.7.7.27] that makes ADP –glucose from glucose-1-P and ATP (the next-tolast step in glycogen synthesis). This was considered a good to mutate since it
will not leave toxic intermediates. The gene was mutated by an in-frame deletion
using a PCR-mediated ligation strategy. The upstream and downstream region
was amplified using primers P1/P2 and P3/P4, respectively. The two PCR
products were combined in a third PCR and the overlapping part introduced by P3
was extended to produce an in-frame truncated NpR6087. The PCR product
containing the deleted gene was re-amplified using P5/P5 primers which
introduced restriction enzymes for cloning into the Km/Nm resistant pRL278.
This strategy was desirable since the start site of the downstream hypothetical
protein lies only 76 bp away and an insertional mutant might show polarity,
causing simultaneous mutation of down stream gene(s).
Location of primers in genome
ATGTTTCTCGCCTCTTCTGCTAAACCTATTGATGACAATCTCAAAAATTTTGTAGAC
GAAATAGAAGCGCAAAGTCCTAGTTTATCAGTTTTAGCAGCGCGTCAATTTCGTTAT
AGTTTGCGTCAAACTACTATAGAAGTAAATATCAAAGAGCCCCGTCAGTTTAACGTA
CTCGAAGAATTTATTATCCGCGCTGCGATTGAATTTCAACCTCCGCCAACAGAGGAT
GAATTAGCCTCTGTACTTGGACTGGATTCTGTATTTATCAAAACTACTACTGCAACT
CTTCGCTCATTACAAACTCTATCAGAAACATCACCAATTACAGTTACTTCTGAAGGT
CGCTCATTTTATGAAAAAGGGTCTGTACCACAGCCCCCATATCCTGTACAGCTTAAT
GCTATTACAGACCCTTTAAGTGACAAAATAACCTTTCAAGCTGAATCACTAAGCGAG
ACAGCGATCAATTTGCCAGACTTGGCAGACTTTTTAACTATTGATCGTGCAATTGCT
GATATCTCTTCTTTAAAACTTGAGGAAATCCAAAAAAGTATTCAGGCTTCAGATTTA
GCACTTCATGTCCCAGAAGAGGGAAAAATCGTTACTTCTTATAAGGTATTAAATCCA
ACTCAAAAATTTTGGAGAAAAATATCGCTTTTCGTTATCTTCGATGCTATTGAAGAT
AAATTAACCATCCAAATAAAAAATGGAAAACAAATTTTAGAGTCAGCATCAAATTGG
TTAGAAGCTCTACACGCTCAAGGGAAGATATCCTTGCAAGCATTATGTAAGTTATCA
ACCGAAACCATTAATTTTGAACGTGAAGCCATTCTCAAATATAAAAATACTGAAATA
GAAGTTAGACTTGAAAATATTCGCCAAAAAGCACTAAAACCTGCGATAAAACCTGGG
AAAAAAGTAGATAATTCTCCAGTCGTAGGTGAAGTTGTACAGTTACGCGATGGACAA
70
ATTAGCCAAGCTTTCTCAGAAGTTTTGAATTCTGCTAAAAGTCAGGTTATTATTTAT
TCTCCTTGGGTGAATCAGGCAGTTGTCGATGAAAAATTTCTAACTTTGTTACAAAAA
TTAGCTAATCGTGGAGTTTGGATTTTAATTGGACATGGAATTGTGCGGCGACAAGAA
GATGAAGAGAAACCAATTTCGCCCGAAGTAGAGAAAAAACTGCGAGCAATAAAAACA
CCTGATGGTTTATCATCTGTACAGGTTTTATGGCTAGGAGACTCCCATATAAAAGAA
GTAATAGTTGATCGAGAAATCCATCTTTGTGGATCTCATAACTGGCTATCTTATCAT
GGTGATTACCTACCACGAGGTGAATCAGTTTATAAAGTCACAATTTTGCATAAAGTC
CAGGAAGCTTATGAATTTCTCGCTAATCGCTTCCAAAATCAAGCTCAAAAATTATGG
CAAAATGCTCTAAACAACCGCGATTCTAAACTGGCTGTGGAGTCTTTATATGTATGG
GGTGCGCTAGGTATGGAAGACATTGCACTCACAAACATACAGCAAAACAATTGGCTA
GAGCTTCTTCCTGTGTGGTTAAAGGTGACACTTCAAGGTTTAAGGTCGAAGAATATA
CAAGCTGATTCAGAAAGTTTTAAAACCGCACTTTCGTTGCTGAGTCAAGTTTCTATT
GAAGAGGCTTTTCTTGAGTCATTGCAGGAAGGATGGCGTAAAGTTATCGGTGCGATC
GCAATTAACAATCCTAAAACTGCTTTAAAGTTACTTAGCGATGAAGTCTGGGCGCAG
TTTCTACGTCTCAATATTGCCCAAAACAGTGATTCACCTGATAATTTTATCTCGCAA
TATACTTTATCAAAGAAAATAAATAGATAActatagcagtcagctttgatttttgtt
cgcacagcgtggcgtagccatatttattggcgtagtgcgtgtagcgcaagcgcgatg
gcaggcaacaggcaagaacgcttttcaagttgtactaaagttttttcagaaatcaaa
tatgattcctatatatcgatttaaatgcaaaagcgatactcaaattccatataaggg
aagtgtagccaaaactttctatattgttcacccacctaaaataatacaaatgttcta
gtctttttacttatcagccgcactatccatcaccaacagcattctataaaattcatc
cctaagaatcacatgatcccccttacctgtggtggcagacagtcctaaactgaagat
gagagttgaaaggcagtcgggagaaattttGTGAAAAAAGTATTATCAATCATCCTT
GGCGGTGGCGCGGGTACTCGGCTTTACCCATTAACCAAACTCCGCGCTAAACCAGCA
GTACCAGTAGCGGGGAAGTACCGCTTAATCGATATCCCTGTTAGTAACTGCATAAAT
TCCGAAATATTCAAAATCTACGTCCTGACACAATTCAACTCAGCTTCTCTGAATCGT
CACATCGCCCGTACCTATAACTTTACTGGCTTTAACGAAGGGTTTGTGGAAGTGCTA
GCGGCACAACAAACTCCAGAAAACCCTAACTGGTTCCAAGGTACAGCAGATGCAGTG
CGTCAGTATCTGTGGTTAATGGAGGAATGGGATGTAGAAGAATATCTAATTCTCTCA
GGCGATCACCTCTACCGCATGGATTATCGCCAGTTTATCCAGCGTCATAGAGATACA
GGAGCTGATATTACTCTCTCAGTTATCCCCATAGACGAGCGCCGTGCCTCAGATTTT
GGTTTGATGAAAATCGATGACTCCGGTAGGATAATTGACTTTAGCGAAAAACCCAAA
GGTGAAGCATTAACCCAAATGCAAGTTGATACAAGTGTACTGGGATTAACAAAAGAA
CAAGCCCAAAAACAACCTTACATCGCTTCGATGGGGATTTATGTCTTTAAAAAAGAG
GTTTTGTTCAAGTTGTTGAGAGAATCTGTAGAACGGACGGATTTTGGTAAAGAAATT
ATTCCTGATGCCTCTAAAGATTACAACGTTCAAGCTTACCTTTTTGATGACTACTGG
GAAGATATTGGAACAATTGAGGCTTTTTATCATGCCAATTTAGCCCTGACTCAGCAG
CCCCAGCCACCCTTTAGTTTCTATGATGAACACGCACCAATTTATACCCGCGCTCGT
TACTTACCTCCGACTAAGCTTTTAGATTGCCAGATCACAGAATCAATTATTGGCGAA
GGTTGTATTTTGAAAAATTGCCGCATTCAACATTCAGTTTTAGGAGTGCGATCGCGA
ATTGAATCTGGTTGCGTCATTGAAGAATCTTTGCTCATGGGTGCAGATTTTTACCAA
GCTTCTGTGGAACGGCAATGTAGCTTGATAGAAAATGACATTCCCGTAGGTATCGGT
ACAGACACGATCATTCGTGGTGCTATCATCGATAAAAATGCGCGCATCGGTCACGAT
GTCAAAATTGTTAATAAAGATAACGTGCAAGAAGCTGAACGCGAAAACCAAGGTTTC
TATATCCGCAGTGGCATCGTTGTCGTGTTGAAAAATGCTGTAATTCCCGATGGAACC
ATCATTTAGtcattagtcattagtcattagtcattggttaatagttacaagtaacaa
aggacaaatgacaaaggacaacggacaaATGACTAAACTCATTTTGTTGATTGGTCT
71
TCCAGGTAGCGGTAAGTCAACTTTTGCAAAAAAATTACTGGTAGAATGCCCCCAGAT
GTCGCTGATTTCTACGGATGCCATCCGGGGGCAATTGTTCGGTTCCCAAGCCCTTCA
GGGACCGTGGGTGCTTATTTGGCACGAAATTGAGCGGCAGTTTCAGCAAGCTATTTC
CAAAACTAATACAGCTATTTTCGATGCTACTAACGCCCAGCGCCGCCATCGTCGTGA
AGTCATTGCTGTAGCACGTAACCTGGGCTTTAGGCAAATTACGGCAATTTGGGTAGA
TACACCAGTCTGGCTGTGTTTAGCATGGAATAAAAAGCGATCGCGCCAGGTTCCTGA
AGAAATTATTTTGCGAATGCACCGTCAACTCCGGGATGCCCCCCCAAGCCTGGAAGA
GGGACTAGACGGGCTGATCCGCTTATCAGAAAAATCGGAGTACGGAAATTGCGATCG
CACGTTGAGCAAGAACCACACTTGAttttctatattctttgttaatttaaaatcaga
ttcttttgcttggcactctacctagcaattaaatatctgtgttcttaaaaaaaaaca
taacagtaatttctataggaggctagtagATGGCTGCAACCGACTTCAAAGACTATT
ACGCAATTTTGGGAGTTAGTAAGACTGCCACTCAGGATGAGATTAAACAAGCCTTTC
GTAAACTAGCCCGCAAATATCACCCCGATGTCAACCCAAATAACAAACAGGCAGAGG
CACGCTTTAAAGAAGTTAGCGAAGCCTACGAGGTTGTTTCAGATGTAGATAAACGCA
AAAAATATGACCAATTCGGCCAATATTGGAAACAAGCTGGTGAAGGTTTCCCCGGCG
GCGGCGTTGGTGCCGATATGGGTGGCTTTGACTTCAGTCAATATGGTAATTTTGATG
AGTTCATTAATGAGTTGCTAGGGCGCTTTGGCGGTGCTGCCCCTCGCGGTGGAGGTG
GGCGACAAAGTTACTCACAAAGTTACTCTTACCGCCCTCCTACAGGTGCGCCAAGTG
GTTTTGGTGGCTTTAACGATTATGGGTTTCAAGATCCAGGTGCGGGTACTTCCCAGG
ATAGTGAAGCTATAATCACCCTAACTTTTGCTGAAGCATTTAATGGCGTGCAAAAGC
GCTTTAGTTTGGGTAACGAGACAATTGATGTTCGTATCCCATCTGGGGCTAAACCTG
GTACTCGTCTGCGCGTGCGGGGTAAAGGTCAAATCAACCCGATGACTCAACAACGAG
GAGATTTATACTTAAAAGTTGAACTTCAGCCGCACTCGTTCTTTCAAATGGAAGGCG
ATAACTTGGTGTGCGAAGTAGCAATTACACCAGATGAAGCTACTCTAGGGGCGTCTA
TTGATGTACCCACTCCCGATGGTTCAGTTAATGTAAAGCTACCAGCCGGAGTGCGTT
CTGGCCAATCGCTGCGTTTGCGTGGCAAAGGTTGGCCCCTCGCCAAGGGTGGACGTG
GCGATCAGTTGGTGAAGGTGGCGATCGCACCACCAAAAGACCTCAGCCAACAAGAAC
GAGAATATTATGAAAAAATCCGGGCTATACGTACTTATAATCCCCGCAGTCATTTGC
AGCAAGTCAAGCTGTGA
The P5/P6 PCR NpR6087deletion product (2,188 base pairs) with restriction
enzyme sites added that was cloned into the sacB plasmid.
GAAGGAGCTCAATTTCTCGCTAATCGCTTCCAAAATCAAGCTCAAAAATTATG
GCAAAATGCTCTAAACAACCGCGATTCTAAACTGGCTGTGGAGTCTTTATATGT
ATGGGGTGCGCTAGGTATGGAAGACATTGCACTCACAAACATACAGCAAAACAA
TTGGCTAGAGCTTCTTCCTGTGTGGTTAAAGGTGACACTTCAAGGTTTAAGGTC
GAAGAATATACAAGCTGATTCAGAAAGTTTTAAAACCGCACTTTCGTTGCTGAG
TCAAGTTTCTATTGAAGAGGCTTTTCTTGAGTCATTGCAGGAAGGATGGCGTAA
AGTTATCGGTGCGATCGCAATTAACAATCCTAAAACTGCTTTAAAGTTACTTAG
CGATGAAGTCTGGGCGCAGTTTCTACGTCTCAATATTGCCCAAAACAGTGATTC
ACCTGATAATTTTATCTCGCAATATACTTTATCAAAGAAAATAAATAGATAAct
atagcagtcagctttgatttttgttcgcacagcgtggcgtagccatatttattg
gcgtagtgcgtgtagcgcaagcgcgatggcaggcaacaggcaagaacgcttttc
aagttgtactaaagttttttcagaaatcaaatatgattcctatatatcgattta
aatgcaaaagcgatactcaaattccatataagggaagtgtagccaaaactttct
atattgttcacccacctaaaataatacaaatgttctagtctttttacttatcag
72
ccgcactatccatcaccaacagcattctataaaattcatccctaagaatcacat
gatcccccttacctgtggtggcagacagtcctaaactgaagatgagagttgaaa
ggcagtcgggagaaattttGTGAAAAAAGTATTATCAATCATCCTTGGCGGTGG
CGCGGGTACTCGGCTTTACCCATTAACCAAACTCCGCGCTAAACCAGCAGTACC
AGTAGCGGGGAAGTACCGCTTAATCGATATCCCTGTTAGTAACTGCATAAATTC
CGAAATATTCAAAATCTACGTCCTGACACAATTCAACTCAGCTTCTCTGAATCG
TCACATCGCCCGTACCTATAATGCTGTAATTCCCGATGGAACCATCATTTAGtc
attagtcattagtcattagtcattggttaatagttacaagtaacaaaggacaaa
tgacaaaggacaacggacaaATGACTAAACTCATTTTGTTGATTGGTCTTCCAG
GTAGCGGTAAGTCAACTTTTGCAAAAAAATTACTGGTAGAATGCCCCCAGATGT
CGCTGATTTCTACGGATGCCATCCGGGGGCAATTGTTCGGTTCCCAAGCCCTTC
AGGGACCGTGGGTGCTTATTTGGCACGAAATTGAGCGGCAGTTTCAGCAAGCTA
TTTCCAAAACTAATACAGCTATTTTCGATGCTACTAACGCCCAGCGCCGCCATC
GTCGTGAAGTCATTGCTGTAGCACGTAACCTGGGCTTTAGGCAAATTACGGCAA
TTTGGGTAGATACACCAGTCTGGCTGTGTTTAGCATGGAATAAAAAGCGATCGC
GCCAGGTTCCTGAAGAAATTATTTTGCGAATGCACCGTCAACTCCGGGATGCCC
CCCCAAGCCTGGAAGAGGGACTAGACGGGCTGATCCGCTTATCAGAAAAATCGG
AGTACGGAAATTGCGATCGCACGTTGAGCAAGAACCACACTTGAttttctatat
tctttgttaatttaaaatcagattcttttgcttggcactctacctagcaattaa
atatctgtgttcttaaaaaaaaacataacagtaatttctataggaggctagtag
ATGGCTGCAACCGACTTCAAAGACTATTACGCAATTTTGGGAGTTAGTAAGACT
GCCACTCAGGATGAGATTAAACAAGCCTTTCGTAAACTAGCCCGCAAATATCAC
CCCGATGTCAACCCAAATAACAAACAGGCAGAGGCACGCTTTAAAGAAGTTAGC
GAAGCCTACGAGGTTGTTTCAGATGTAGATAAACGCAAAAAATATGACCAATTC
GGCCAATATTGGAAACAAGCTGGTGAAGGTTTCCCCGGCGGCGGCGTTGGTGCC
GATATGGGTGGCTTTGACTTCAGTCAATATGGTAATTTTGATGAGTTCATTAAT
GAGTTGCTAGGGCGCTTTGACTAGTCTGC
73
Cyanophycin Mutagenesis
NpR5823 (cyanophycin synthetase) in blue and ~1500 bp on either side. Red text
denotes R5824 encoding cyanophycinase involved in degradation of this polymer.
Strategy is similar to that above.
Cyanophycin mutagenesis primers
P1
GGACACCCCAAGCAACTAAA
P2
GGCGTCGAATGCTCCAATAGT
P3
ACTATTGGAGCATTCGACGCCCTCAAAATGGGATTGCACCT
P4
CCGAGATTTTCGTGATTGGT
P5 (Xho1)
TACTCGAGCGGGTTTGGGATTTATTCCT
P6 (BamH1)
TTGGATCCTGGAATGCTGAAGGCTTCTT
location of primers in genome
aagtggacaggtgaaaaacaaggccctggctttgaggaatgggaacaaacacgt
ttagactgaggcaacgcgagcgttaagcgagaattcggagagtggctgcccagt
ttcaaaacttccacaaagtgtcctctcgaaagttgcaaagcgcaaaaatcatca
gccttctgaaccttgttaaattcacctgaagttgttgacaggagaaacaaaatg
ccgcaattacaagctaaatcgctagaaatgaggacaccccaagcaactaaaacc
gccgttctggttatcggaggcgcagaagacaaagttcatgggcgcgaaatccta
cgaactttttttggacgtgccggtgctagtaaggcttatattacaattattcca
tctgcttctcgcgaacctgccatcattggtggtcggtacattcgcatttttgaa
gaaatgggtgcccaaaaggtagagattttagacatccgcgaacgcgaacagtgt
gaagcctctcaaatcaaagcatccttagaagcctgtagtggggtatttttgaca
ggaggagaccaactccgtctctgtggtgtattggcagatacgccagcaatggaa
attattcggcagagggttagggcagggcaactgacgttagcaggcaccagtgcc
ggagcggcagtgatggggcatcacatgattgctggcggcgggagtggagagtcg
ccaaatcgttccctagtagatatggcaacgggtttgggatttattcctgaagtc
atcgttgaccaacattttcacaaccggaatcggatggggcggttgattagtgcg
atcgctgctcaccccgatcgcttaggtattggcattgacgaagatacttgtgca
gtgtttgaacgtgatggttggttacaagttatgggtaaaggcagtgttaccatt
gtcgatcccactgaagccacccacaccaacgaaccccatgttggtgctaatgaa
ccattaaccgtgcataatttacgtctccatatcctcagctacggcgatcgcttc
cacctgtaccagcgcactgtattgcctgctgtacaccggatctccagctgacgg
gatagagtatctgaggttacactgagttgaccacaaattttagattttggattt
tgcgaaaagttgtaaggagggtttcccgacctagcaaacttttcaagacggatt
ttagattaagagagcttttcggcagatgccgccagaaagtgggtaaaacaaatc
taaaaacccctcgatgactcgttgacgctacgctattgtaaatctaaaattacc
agagctaagtaccctaattgtctcttttaatccaaaatcataaatctaaaatcc
aaaatcggttgatcgcacatttatttcttgttgtagaaaaatgatgagaatact
atgtaaaaagaaaaaactgtttgcaatgaacagtttagcggtcaattacagtaa
gaaactagaattttggttccgaatctccatctacctattcccATGAGAATCCTC
AAGATCCAGACCTTACGCGGCCCAAACTATTGGAGCATTCGACGCCACAAGCTG
ATCGTCATGCGCCTCGATTTAGAAACCCTTGCCGAGACGCCCTCGAATGAAATC
CCAGGCTTTTATGAAGGACTAGTTGAGGCGCTGCCGAGTCTGGAGGGTCATTAT
TGTTCGCCTGGCTGTCGTGGTGGTTTTTTGATGCGAGTCAAAGAAGGCACGATG
74
ATCGGCCATATCGTAGAACACGTAGCCTTAGAACTCCAGGAATTAGCTGGTATG
CACGTCGGCTTTGGTCGCACCCGCGAAACTGCCACACCCGGAATTTATCAAGTA
GTAATTGAATACCAGAATGAGGAAGCGGGACGCTACGCCGGACGAGCCGCAGTG
CGGCTGTGCCAGAGTATCGTCGATCGAGGCCGTTATCCCAAGGCAGAACTAGAG
CAAGATATCCAAGACCTGAAAGACTTCACCCGTGATGCTTCTTTAGGCCCTTCT
ACTGAAGCAATCATCAAAGAAGCAGAAAAAAGAGGTATTCCCTGGATGCCTCTG
GAAGCCCGCTTTTTGATTCAGCTAGGCTACGGCGTGAACCAGAAGCGGATGCAA
GCCACAATGACTGACAATACCAGCATTCTGGGCGTAGAACTAGCTTGCGACAAA
GAAGCCACAAAACGTATCCTGGCTGCGGCTGGTGCGCCAGTACCCAGAGGCACA
GTAATCAACTTCTTAGACGATTTGGAACAAGCTATTGAATACGTTGGCGGCTAT
CCCATCGTCGTCAAGCCCCTGGATGGCAATCATGGACGTGGGATCACCATTGAC
ATCAGAACTTGGGAAGAAGCGGAAGCTGCCTATGAAGCCGCTAGACAGGTTTCC
CGGTCAATTATCGTCGAAAGATATTACGTTGGGCGTGACCACAGGGTACTAGTG
GTAAATGGCAAAGTAGTCGCGGTAGCTGAACGTGTCCCAGCTCACGTCATTGGC
AATGGCAGATCGACCATCTCCGAACTGATTGAGGAAACAAACCTCGATCCAAAT
CGCGGTGAAGGACATGATAACGTCCTGACCAAAATTGAACTAGATCGCACCAGC
TATCAACTATTAGAAAGGCAAGGCTATACCCTAAATAGCGTTCCACCCAAGGGT
ACTATTTGTTATCTCAGGGCAACAGCCAACTTAAGTACAGGTGGTAGCGCCGTA
GATCGTACCGATGAAATTCACCCAGAAAATCTGTGGTTGGCACAACGAGTAGTC
AAGATTATCGGTTTAGATATCGCCGGACTCGATATCGTCACCACGGATATTAGC
CGTCCCCTGCGGGAAGTCGATGGCGTAATTGTTGAAGTTAACGCCGCTCCCGGC
TTCCGGATGCACGTTGCGCCAAGCGTGGGTATTCCCCGCAACGTCGCTGGTGCA
GTGATGGATATGCTGTTCCCTAACGAACAATCTAGCCAAATTCCTATCCTCAGT
GTTACTGGTACTAATGGCAAAACCACTACTACCCGGCTATTAGCACATATTTAT
AAACAGACTGGTAAAGTAGTCGGATATACTACAACCGATGGAACATACATCGGC
GATTACTTAGTGGAAGCTGGCGATAACACAGGCCCACAAAGTGCCCATGTCATT
CTCCAAGATCCCACAGTAGAAGTAGCAGTACTGGAAACCGCCCGCGGTGGCATT
CTCCGCTCTGGATTGGGCTTTGAAGCCGCAAATGTAGGAGTGGTATTAAATGTA
GCCGCCGACCACTTAGGAATAGGCGATATAGAGACTATTGAGCAATTAGCTAAC
CTCAAGAGTGTAGTAGCGGAAGCCGTATTCCCTGATGGCTACGCGGTACTTAAC
GCCGACGATCATCGCGTCGCCGCTATGTCAGAAAAAACTAAGGCTAATATTGCT
TACTTCACCATGAATCCCGACTCGGAATTGGTGCGAAAGCACATCCAAAAGGGT
GGAGTAGCCGCAGTCTATGAAAGCGGCTATTTGTCAATTGTTAAAGGTGATTGG
ACACATCGGATAGAAAGAGCAGAAAATATACCTTTAACAATGGGCGGACGTGCG
CCGTTTATGATTGCCAACGCTTTAGCTGCAAGTTTGGCAGCATTCGTGCAAAAC
GTCACGATTGAGCAGATTCGCTCTGGTTTGAAGACTTTCCGAGCTTCAGTTAGT
CAAACGCCGGGACGAATGAATTTATTTAATTTAGGCAACTACCACGCTTTAGTA
GACTATGCTCACAACGCAGCTAGTTATGAAGCTGTAGGTTCCTTTGTACGGAAC
TGGACTACAGGACAACGGATTGGCGTAATTGGTGGCCCGGGCGATCGCCGCGAC
GAAGATTTTGTTACTTTGGGCAAATTAGCAGCACAAATTTTTGACTACATCATT
GTCAAAGAAGACGATGATACACGGGGACGTTTACGGGGTTCAGCCGCCCAATTA
ATTATTCAAGGCATTACCCAAGTGAAGCCTGATAGCCGCTATGAATCAATTTTG
GATGAAACCCAAGCGATTAATAAAGGCTTAGACATGGCTCCTGATAACAGCCTG
GTGGTGATTCTACCAGAAAGCGTGACTAGGGCTATTAAGTTAATTAAGCTGCGT
GGTTTAGCCAAGGAAGAGACACATCAACAAAATACTGGCACAGCGATCGCTGAT
TCTCAAAATGGGATTGCACCTTCTTCTGTAGTCAACACACTACTGTAGtcaaca
gtcaagagtctcaagttcagcatttcaaagtttttcagccttgactcttgacta
75
ttgactattgtttttcttcttctgaattagtttcatcactgggggttttcatct
cctccttaaaacctcgtagggttttgcctagtgcagtgcccaattcgggaattt
ttttagggccaaaaattagaatagcgactatggtaattacagcgatttctggcc
atcctagtccaaacatataaatttcctctaaggcatctctaattaaatatagac
attgatggctcaaatctgatgatttaggggcaaacaagaggtaaggaagtaggg
agtaaggggaatagttcaagcaacgattgaccccgcttgttgctctagtggtct
gtcccattaaatttgattcgcctatcaaatttaaatttctcttctttccttctt
cctctgcgctctacccttcgggaagccacttctctatgagacgctccgcgaacg
tgtctatgcgtccttctctaacgagacactgcgcgtagcttgcttcaccgtagg
agtatgcggttcgttctcttatagtcatttccattgaatcagggaagattacgc
taggggttgcgcgtgaggacagagaaactgcgtaaagcttcctattatttttga
agttaaaagataagcttatctttcttttgcttagaatctaaaggtatcaattca
accaaggcgaagggaatgatacatagagcccataagaaatattgtaagaataat
actgaattaaaatacaaaaatatcatcataattattgtggaagatacaaacata
ctcaccttttctttcaggaaacttaggtaaagcattcccatcagaaatagcatg
agaaacttggctcttaatcctacaatccaggtgaatttattagaaaaaataaca
tctacagagggtgcagcccctatatgcaaactttctaaacgtgttgcagaaaac
aagatggatttgaagaagccttcagcattccaaatcagaaaaggtaatgatgtt
aaaagaggaatgcttaaaataataaaaaatccccaaactaaatttttaccccgc
ttatttcctactccatttttccataagtagatgagatacaaaggcagcaggaat
attgcaatctgtttaattcccaaagagatactaaacataagtaaagaaagttta
gtcttctctttcaaaaggactaaagacaatagtaaaaaaaatatcgctgcaaat
tccagatggtgaacacgtactatatagatactccagcgacctaataaaagaatt
gtagaggctacaaagccaagtatccataaacctcgtatctggaaatatctcaat
gttaaggcaacgataccaatatggaaggcaaaagaaataggtcgccacaaatat
aaccaatcacgaaaatctcggaaaccaagcttttgaactagcgcagagaaaagg
taggcaagtgggaaataagttgcatatttatcattg
The P1/P4 PCR product (2788 base pairs) with NpR5823 (cyanophycin
synthetase) in-frame gene deletion
ggacaccccaagcaactaaaaccgccgttctggttatcggaggcgcagaagaca
aagttcatgggcgcgaaatcctacgaactttttttggacgtgccggtgctagta
aggcttatattacaattattccatctgcttctcgcgaacctgccatcattggtg
gtcggtacattcgcatttttgaagaaatgggtgcccaaaaggtagagattttag
acatccgcgaacgcgaacagtgtgaagcctctcaaatcaaagcatccttagaag
cctgtagtggggtatttttgacaggaggagaccaactccgtctctgtggtgtat
tggcagatacgccagcaatggaaattattcggcagagggttagggcagggcaac
tgacgttagcaggcaccagtgccggagcggcagtgatggggcatcacatgattg
ctggcggcgggagtggagagtcgccaaatcgttccctagtagatatggcaacgg
gtttgggatttattcctgaagtcatcgttgaccaacattttcacaaccggaatc
ggatggggcggttgattagtgcgatcgctgctcaccccgatcgcttaggtattg
gcattgacgaagatacttgtgcagtgtttgaacgtgatggttggttacaagtta
tgggtaaaggcagtgttaccattgtcgatcccactgaagccacccacaccaacg
aaccccatgttggtgctaatgaaccattaaccgtgcataatttacgtctccata
tcctcagctacggcgatcgcttccacctgtaccagcgcactgtattgcctgctg
tacaccggatctccagctgacgggatagagtatctgaggttacactgagttgac
cacaaattttagattttggattttgcgaaaagttgtaaggagggtttcccgacc
76
tagcaaacttttcaagacggattttagattaagagagcttttcggcagatgccg
ccagaaagtgggtaaaacaaatctaaaaacccctcgatgactcgttgacgctac
gctattgtaaatctaaaattaccagagctaagtaccctaattgtctcttttaat
ccaaaatcataaatctaaaatccaaaatcggttgatcgcacatttatttcttgt
tgtagaaaaatgatgagaatactatgtaaaaagaaaaaactgtttgcaatgaac
agtttagcggtcaattacagtaagaaactagaattttggttccgaatctccatc
tacctattcccATGAGAATCCTCAAGATCCAGACCTTACGCGGCCCAAACTATT
GGAGCATTCGACGCCTCAAAATGGGATTGCACCTTCTTCTGTAGTCAACACACT
ACTGTAGtcaacagtcaagagtctcaagttcagcatttcaaagtttttcagcct
tgactcttgactattgactattgtttttcttcttctgaattagtttcatcactg
ggggttttcatctcctccttaaaacctcgtagggttttgcctagtgcagtgccc
aattcgggaatttttttagggccaaaaattagaatagcgactatggtaattaca
gcgatttctggccatcctagtccaaacatataaatttcctctaaggcatctcta
attaaatatagacattgatggctcaaatctgatgatttaggggcaaacaagagg
taaggaagtagggagtaaggggaatagttcaagcaacgattgaccccgcttgtt
gctctagtggtctgtcccattaaatttgattcgcctatcaaatttaaatttctc
ttctttccttcttcctctgcgctctacccttcgggaagccacttctctatgaga
cgctccgcgaacgtgtctatgcgtccttctctaacgagacactgcgcgtagctt
gcttcaccgtaggagtatgcggttcgttctcttatagtcatttccattgaatca
gggaagattacgctaggggttgcgcgtgaggacagagaaactgcgtaaagcttc
ctattatttttgaagttaaaagataagcttatctttcttttgcttagaatctaa
aggtatcaattcaaccaaggcgaagggaatgatacatagagcccataagaaata
ttgtaagaataatactgaattaaaatacaaaaatatcatcataattattgtgga
agatacaaacatactcaccttttctttcaggaaacttaggtaaagcattcccat
cagaaatagcatgagaaacttggctcttaatcctacaatccaggtgaatttatt
agaaaaaataacatctacagagggtgcagcccctatatgcaaactttctaaacg
tgttgcagaaaacaagatggatttgaagaagccttcagcattccaaatcagaaa
aggtaatgatgttaaaagaggaatgcttaaaataataaaaaatccccaaactaa
atttttaccccgcttatttcctactccatttttccataagtagatgagatacaa
aggcagcaggaatattgcaatctgtttaattcccaaagagatactaaacataag
taaagaaagtttagtcttctctttcaaaaggactaaagacaatagtaaaaaaaa
tatcgctgcaaattccagatggtgaacacgtactatatagatactccagcgacc
taataaaagaattgtagaggctacaaagccaagtatccataaacctcgtatctg
gaaatatctcaatgttaaggcaacgataccaatatggaaggcaaaagaaatagg
tcgccacaaatataaccaatcacgaaaatctcgg
The P5/P6 PCR product (1884 base pairs) with NpR5823 (cyanophycin
synthetase) in-frame gene deletion and restriction enzyme sites added by these
primers.
TACTCGAGcgggtttgggatttattcctgaagtcatcgttgaccaacattttca
caaccggaatcggatggggcggttgattagtgcgatcgctgctcaccccgatcg
cttaggtattggcattgacgaagatacttgtgcagtgtttgaacgtgatggttg
gttacaagttatgggtaaaggcagtgttaccattgtcgatcccactgaagccac
ccacaccaacgaaccccatgttggtgctaatgaaccattaaccgtgcataattt
acgtctccatatcctcagctacggcgatcgcttccacctgtaccagcgcactgt
attgcctgctgtacaccggatctccagctgacgggatagagtatctgaggttac
actgagttgaccacaaattttagattttggattttgcgaaaagttgtaaggagg
77
gtttcccgacctagcaaacttttcaagacggattttagattaagagagcttttc
ggcagatgccgccagaaagtgggtaaaacaaatctaaaaacccctcgatgactc
gttgacgctacgctattgtaaatctaaaattaccagagctaagtaccctaattg
tctcttttaatccaaaatcataaatctaaaatccaaaatcggttgatcgcacat
ttatttcttgttgtagaaaaatgatgagaatactatgtaaaaagaaaaaactgt
ttgcaatgaacagtttagcggtcaattacagtaagaaactagaattttggttcc
gaatctccatctacctattcccATGAGAATCCTCAAGATCCAGACCTTACGCGG
CCCAAACTATTGGAGCATTCGACGCCTCAAAATGGGATTGCACCTTCTTCTGTA
GTCAACACACTACTGTAGtcaacagtcaagagtctcaagttcagcatttcaaag
tttttcagccttgactcttgactattgactattgtttttcttcttctgaattag
tttcatcactgggggttttcatctcctccttaaaacctcgtagggttttgccta
gtgcagtgcccaattcgggaatttttttagggccaaaaattagaatagcgacta
tggtaattacagcgatttctggccatcctagtccaaacatataaatttcctcta
aggcatctctaattaaatatagacattgatggctcaaatctgatgatttagggg
caaacaagaggtaaggaagtagggagtaaggggaatagttcaagcaacgattga
ccccgcttgttgctctagtggtctgtcccattaaatttgattcgcctatcaaat
ttaaatttctcttctttccttcttcctctgcgctctacccttcgggaagccact
tctctatgagacgctccgcgaacgtgtctatgcgtccttctctaacgagacact
gcgcgtagcttgcttcaccgtaggagtatgcggttcgttctcttatagtcattt
ccattgaatcagggaagattacgctaggggttgcgcgtgaggacagagaaactg
cgtaaagcttcctattatttttgaagttaaaagataagcttatctttcttttgc
ttagaatctaaaggtatcaattcaaccaaggcgaagggaatgatacatagagcc
cataagaaatattgtaagaataatactgaattaaaatacaaaaatatcatcata
attattgtggaagatacaaacatactcaccttttctttcaggaaacttaggtaa
agcattcccatcagaaatagcatgagaaacttggctcttaatcctacaatccag
gtgaatttattagaaaaaataacatctacagagggtgcagcccctatatgcaaa
ctttctaaacgtgttgcagaaaacaagatggatttgaagaagccttcagcattc
caGGATCCaa
78