The 8th
Annual
NC
Undergraduate
Summer Research Symposium
Synthetic Biology
Research Experience for Undergraduates abstracts
Abstracts are listed in
alphabetical order by the last name of the corresponding author.
Blumert, Jacob F. |
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Home
Institution: |
Manhattan College |
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Program: |
Synthetic
Biology Research Experience for Undergraduates |
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College: |
CALS |
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Department(s): |
Plant Biology |
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Research
Mentor(s): |
Wendy Boss/Plant Biology Yang Ju Im/Plant Biology |
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Title of
Presentation: |
Are Stress Tolerant Tomatoes a Possibility |
Ever
since the end of nomadic society and the dawn of civilization, people have relied
on agriculture. As populations increased, scientists have developed enhanced
disease resistant crops. Recently we have been reaching the limits of improved
crop productivity resulting from the “Green Revolution.” To address these
challenges we have taken a synthetic approach to generating more tolerant
plants. When plants become stressed they
produce superoxides and other reactive oxygen species as chemical response
signals. In extreme stress conditions, excess amounts of reactive oxygen
species will inhibit plant growth and, if endured long enough, cause
fatality. Pyrococcus furiosus is an
Archaea that has an efficient mechanism to reduce with reactive oxygen species,
specifically superoxide. Our hypothesis is introducing the Superoxide Reductase
(SOR) pathway, found in P. furiosus, into tomatoes
would make them more stress tolerant and therefore more productive. In this
research project, I test the putative transgenic tomato plants to determine if
the plants had the SOR gene inserted into their genome. The second aspect of
the project was to establish that the gene was being transcribed and mRNA
propagated. The last and most pertinent portion of the project was to show that
SOR proteins exist throughout the plant and the plant was more stress tolerant. From this project I have determined that
several transgenic tomato lines have acquired the P. furiosus SOR gene,
transcribed it, translated it and produce the P. furiosus SOR proteins.
Brown, Erica M. |
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Home Institution: |
NCSU |
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Program: |
Synthetic
Biology Research Experience for Undergraduates |
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College: |
CALS |
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Department(s): |
Plant Biology |
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Research
Mentor(s): |
Heike Winter Sederoff/Plant Biology Qian Wu/Plant Biology Marie-Laure Sauer/Plant Biology |
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Title of
Presentation: |
How Do Plant Roots Sense Mechanical Stress? |
The
sensitivity of a root’s mechanical sensory and response system is detrimental to
the sustainability of sessile plants in ever-changing environmental
conditions. Our hypothesis is that a
defect in the plant roots sensing/response system to mechanical impedance will
cause the root to grow into medium impenetrable to wild type plants. First we determined an effective agar
concentration for mechano-insensitive screening. Insensitive mutants (containing T-DNA
insertions) were identified by their lack of avoidance resulting in growing
into the dense medium avoided by wild type.
We plan to perform a secondary screening to confirm positive
mutants. Following identification, we
plan to grow the insensitive mutants, gather their offspring, and perform DNA
extractions and TAIL-PCR. The TAIL-PCR
will help isolate the location of T-DNA insertions and identify the genes
involved in mechano-sensing of plants.
From that point we will attempt to map out a pathway by which events
occur to make mechano-sensing possible.
Chisnell, Peter J. Marrs, Julia K. |
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Home
Institution: |
Vanderbilt University Barnard College of Columbia University |
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Program: |
Synthetic
Biology Research Experience for Undergraduates |
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College: |
CALS |
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Department(s): |
Microbiology |
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Research
Mentor(s): |
Amy M. Grunden/Microbiology YangJu Im/Microbiology Wendy F Boss/Plant Biology |
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Title of
Presentation: |
Confirmation of Expression and Activity of an Archaeal Superoxide Reductase
(SOR) Gene in Transgenic Tomato Lines Developed for Enhanced Stress
Resistance |
Creating
stress resistant crops has always been a priority for researchers in the
agricultural field. Plants that can weather drought, heat, and other stressors
without losing yield can benefit many human projects including food and fuel
production. Such stress resistance has been obtained in the model plant
Arabidopsis by engineering it to express superoxide reductase (SOR), an enzyme
found in the hyperthermophilic archaeon Pyrococcus furiosus. The goal of
our work is to determine if this stress resistance can be expressed in a crop
plant, specifically tomato (Solanum lycopersicum) cv. ‘Micro Tom’.
Studies were conducted with a transgenic tomato line shown to transcribe mRNA
from the SOR gene. SOR-expressing tomato displayed a phenotype different from
wild type, with fruits that change slowly in color from green to yellow to red,
as opposed to the wild type phenotype of fruits which change directly from
green to red. We hypothesize that this phenotypic difference is due to
SOR inhibiting the action of reactive oxygen species (ROS) required to convert
pro-lycopene (yellow) in the fruit to lycopene (red). First, in order to
isolate the SOR protein from tomato tissue, an immunoprecipitation was
conducted. Western blot analysis was then performed and confirmed the
presence of the SOR protein inside the tomato tissues. A WST-SOD assay
was then conducted to measure the ability of SOR to degrade superoxide.
The assay quantifies the amount of a colored formazin compound that is
generated in response to O2¯; higher SOR activity results in lower production
of this compound. Results show an increased ability in the transgenic
line to quench superoxide compared to wild type plants. This work
confirmed the presence of the P. furiosus SOR protein in transgenic tomato and
its activity in reducing ROS.
Estes, Christian S. |
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Home
Institution: |
NCSU |
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Program: |
Synthetic
Biology Research Experience for Undergraduates |
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College: |
Engineering and Technology |
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Department(s): |
Plant Biology |
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Research
Mentor(s): |
Imara Perera/Plant Biology Wendy Boss/Plant Biology |
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Title of
Presentation: |
Is InsP3-mediated Signaling Involved in
the Plant Innate Immunity Response? |
In the present climate, rising temperatures could
lead to more frequent and longer periods of drought in areas that previously
had never experienced these types of weather. It is essential, to find ways to
modify plant life in order to cope with these circumstances for food sources to
continue growing efficiently and without hindrance. A transgenic line of
Arabidopsis thaliana has been developed that expresses the mammalian type I
inositol polyphosphate 5-phosphatase (InsP5-ptase) and that shows an increased
tolerance to drought conditions. Plants expressing this transgene have been
found to develop normally under regular conditions but have been found to have
increased susceptibility to bacterial infection. The purpose of this experiment
is to determine whether this sensitivity to bacteria comes from a downstream
change to the Innate Immunity pathway. The Innate Immune pathway is an elicitor-induced
pathway, which recognizes pathogen associated molecular patterns. In order to
determine if this pathway is impaired, we treated Arabidopsis seedlings with a
peptide derived from the bacterial elicitor flagellin (flg 22). Samples were
harvested at different times after treatment and RNA was isolated. We compared
expression patterns between wild type Arabidopsis and the transgenic line.
Several flagellin-induced genes such as WRKY 29 and MLO6 were amplified using
Quantitative Real Time PCR (qPCR) and Actin was used as a control. To look for
comparative changes in their root growth, seedlings were grown in the presence
and absence of flg 22. After a period of five days, the roots were then
measured and lengths were compared. Our results will reveal if plant innate
immunity is compromised in the transgenic plants.
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Hernández, Liz J. |
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Home
Institution: |
University of Puerto Rico at Río Piedras |
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Program: |
Synthetic
Biology Research Experience for Undergraduates |
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College: |
CALS |
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Department(s): |
Plant Pathology |
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Research
Mentor(s): |
Gary A. Payne/Plant Pathology Andrea L. Dolezal/Plant Pathology Greg OBrian/Plant Pathology |
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Title of
Presentation: |
Characterization of Role of aflpesA, a Gene Involved in Secondary Metabolism of Aspergillus flavus |
The ability of fungi to resist reactive oxygen
species (ROS) produced by the host is important for pathogenicity. The gene pesA that confers tolerance to ROS in Aspergillus fumigatus is required for
full pathogenicity. The objective of
this study was to mutate the homologue of pesA
(aflpesA) in A. flavus and
determine if the mutants are impaired for pathogenicity on Arabidopsis. To mutate aflpesA
in A. flavus, we identified the AMP-binding
domain of the 700 kDa aflPesA protein
and targeted it for replacement with argD,
a selectable marker for fungal transformation. Generation of the disruption
construct is a two step process. In the
first PCR reaction, we generated the expected 1.7 kb of 5’ and 1.5 kb of 3’
flanking regions of the domain and the 2.2 kb argD fragment. These
fragments were either gel purified, column purified or used without further
cleanup in subsequent steps. For the second step, the three fragments were
combined and used in an overlap PCR protocol to generate the disruption
construct. PCR products were gel
purified and cloned into TOPO 2.1.
Bacterial transformants are currently being screened by PCR in order to
identify colonies harboring the disruption construct. In a related part of this project we are
confirming the reliability of Arabidopsis as a screen to evaluate mutants of A. flavus impaired in
pathogenicity. Wild type A. flavus and a mutant, sodA, which is impaired in its ability
to tolerate ROS, are being tested on wild type Arabidopsis and two mutants will
altered production of ROS. Results from these studies will provide
further insight into the role of fungal secondary metabolites in fungi.
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Leonardo, Tracey |
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Home Institution: |
Columbia University |
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Program: |
Synthetic
Biology Research Experience for Undergraduates |
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College: |
CALS |
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Department(s): |
Plant Biology |
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Research
Mentor(s): |
Marcela Rojas-Pierce/Plant Biology |
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Title of
Presentation: |
Protein Trafficking and Responses to Auxin and Salt
Stress in gravacin-hypersensitive 2
Mutants |
Endomembrane trafficking is known to play roles in
nearly all biological processes in plant cells–from cell signaling to cell wall formation. The genes that encode parts
of this system are therefore of high interest, especially because little is
known about them. To study these genes and avoid the lethality associated with
classical mutant screens, chemical genomics was therefore used. Gravacin is a
small chemical that inhibits the localization of d-TIP, a transmembrane protein
of the tonoplast. An Arabidopsis thaliana
mutant (grav-hs2) was previously
identified to be hypersensitive to gravacin at a 2.5uM because it showed
shorter roots as compared to the wild type. Three putative T-DNA insertions
were then identified in grv-hs2. The
goals of this project were therefore to: (1) Identify the gene(s) responsible
for the short root phenotype in grav-hs2;
2) To determine if grav-hs2 has any
phenotypes in trafficking; and (3) Identify any phenotype in this mutant
associated with gravitropism and/or the auxin and salt stress responses.
Results here show that a T-DNA insertion is present between At4g25600 and At4g25610,
but no changes in the expression of these genes were detected. Furthermore, grv-hs2 showed mis-localization of
GFP-d-TIP at low concentrations of Gravacin, indicating a role of the tagged
gene in protein trafficking. The response of grv-hs2 to changes in gravitropism, auxin and salt stress signals
were normal, indicating that the phenotype may be specific to trafficking
pathways. Future research on the gene responsible for Gravacin-induced
mis-localization of d-TIP in these mutants will provide more information about
the biogenesis of the tonoplast.
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Marrs, Julia K. Chisnell, Peter J. |
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Home
Institution: |
Barnard College of
Columbia University Vanderbilt University |
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Program: |
Synthetic
Biology Research Experience for Undergraduates |
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College: |
CALS |
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Department(s): |
Microbiology |
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Research
Mentor(s): |
Amy M. Grunden/Microbiology Wendy F. Boss/Plant Biology Yangju Im/Plant Biology |
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Title of
Presentation: |
Does Fusion with Green Fluorescent Protein Make
Superoxide Reductase a Better Antioxidant Enzyme? |
Previously, an Arabidopsis model of plant stress
resistance was designed by transforming the antioxidant gene superoxide
reductase (SOR) from the archaeal hyperthermophile Pyrococcus furiosus into Arabidopsis. The SOR gene was inserted into the
Arabidopsis genome as a fusion with green fluorescent protein (GFP) to provide
information about its expression and localization in the plant. A new study suggested that GFP acts as an
electron donor, which could affect the activity of transgenic GFP-SOR since SOR
reduces superoxide by a one electron transfer process. Recently SOR has been transformed into tomato
as a single gene (not as part of a GFP fusion).
Therefore, in light of the new GFP findings and our different
Arabidopsis and tomato SOR transgenic plants (GFP-fused versus single gene), we
are interested in determining whether GFP, when fused with SOR, influences the
activity of SOR. To investigate differences in SOR and GFP-SOR activity, we
have attempted to clone GFP-SOR into a pTrc99A expression vector for
over-expression in Escherichia coli.
The resulting expression plasmid was transferred into the superoxide dismutase
(SOD) mutant E. coli strain, NC906
and activity assays were used to measure cytochrome c inhibition activity of
GFP-SOR protein products in comparison with SOR alone. Half-life experiments were also performed on
GFP-SOR and SOR extracts in order to compare their stability. Results of these activity assays will be
presented as an examination of the effect of GFP on SOR activity, so that the
function of GFP-SOR or SOR within transgenic plants can be better understood. Additionally, this characterization will aid
in the effort to produce reliably stress resistant crop plants.
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Meador, Lydia R. |
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Home
Institution: |
Oklahoma State University |
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Program: |
Synthetic
Biology Research Experience for Undergraduates |
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College: |
CALS |
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Department(s): |
Botany |
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Research
Mentor(s): |
De-Yu Xie/Plant Biology Patrick T. Walker/Plant Biology Ming-Zhu Shi/Plant Biology |
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Title of
Presentation: |
Characterization of PAP1-transgenic Cell Suspension
Culture Through Growth, Anthocyanins, and Gene Expression |
Anthocyanins are plant flavonoid pigments providing
pink/red/blue/purple coloration in most plants. They attract both pollinators
and seed dispersers. In addition, their
antioxidative activity protects plants from radiation-induced damage on
cells. Furthermore, their presence in
food provides strong antioxidative uses benefiting human health. PAP1 is one of the R2R3-MYB transcription
factors directly regulating the biosynthesis of anthocyanins. We have established PAP1-transgenic callus
and suspension cell cultures of tobacco. In this study, we characterize the
growth of cells, anthocyanin formation, and gene expression in
suspension-cultured transgenic red cells (6R) of PAP1 during a 25-day culture
period. Transgenic white cells (6W) of PAP1 and wild-type cells (P3) were used
as controls. Samples were collected from six time points at days 0, 5, 10, 15,
20, and 25. Growth curves for the change in fresh weight were measured for these
suspension culture cells. The biomass increase of the 6R cells followed a
sigmoid-like trend. The biomass increase of the P3 cells followed a logarithmic
trend. The fresh weight increase of the
P3 cells was faster than that of the 6R cells. A dynamic trend of anthocyanin
levels were observed in the 6R cells. The P3 cells did not produce
anthocyanins. In addition, we will present and discuss the anthocyanin
structures and profiles and the expression levels of the PAP1 transgene and its
targeted pathway genes (e.g. PAL, CHS, DFR, and ANS). This research is funded USDA-NRI (proposal
number 2006-1334).
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Miguel, Lourdes A. |
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Home
Institution: |
University of Puerto Rico at Mayaguez |
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Program: |
Synthetic
Biology Research Experience for Undergraduates |
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College: |
Engineering and Technology |
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Department(s): |
Chemical and Biomolecular Engineering |
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Research
Mentor(s): |
Robert M. Kelly/Chemical and Biomolecular Engineering Sara Blumer-Schuette/Chemical and Biomolecular Engineering Inci Ozdemir/Chemical and Biomolecular Engineering |
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Title of
Presentation: |
Identification and Characterization of Cellulases from Extreme
Thermophiles for Biomass Deconstruction |
Extremely
thermophilic bacteria, some of which have cellulose degrading enzymes, have the
potential to use plant biomass as a substrate, yielding hydrogen and ethanol as
products. This characteristic makes them a valuable prospective target of
research, for the production of biofuels. Concentrations of biomass substrate
(switchgrass) that Caldicellulosiruptor
saccharolyticus can tolerate are being investigated, in addition to the
time that it takes for C. saccharolyticus
to adapt to each increased concentration of substrate. With the genome of C. saccharolyticus previously sequenced,
the designed oligonucleotide microarray can be used to identify different genes
that are expressed for adaptation to a specific concentration of substrates
such as switchgrass and crystalline cellulose. Currently C. saccharolyticus tolerated a concentration of 12 g/L switchgrass
after five passages, and adaptation to growth at 16 g/L switchgrass is
currently ongoing. In addition, individual cellulase enzymes of C. saccharolyticus: Csac_0678, Csac_1080
and Csac_0137 were characterized referent to their optimum temperature, pH and
enzymatic half life. These recombinant enzymes were produced and purified by
using FPLC. Reducing sugar production was monitored by a DNS reaction. So far,
thermophilic cellulases have been reported as having an optimum temperature of
50°C an optimal pH of 5.0 with a half
life of 16 min-1; but since C.
saccharolyticus has an optimum growth temperature of 70°C, its cellulases may have an optimum temperature as
high as 65-80°C. The ability of C. saccharolyticus to hydrolyze cellulose at higher temperatures
and to tolerate increased solids in growth media makes it a valuable prospect
for next generation biofuels production.
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Murphree, Colin A. |
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Home
Institution: |
Transylvania University |
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Program: |
Synthetic
Biology Research Experience for Undergraduates |
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College: |
CALS |
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Department(s): |
Botany |
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Research
Mentor(s): |
Niki Robertson/Botany Rich Tuttle/Botany |
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Title of
Presentation: |
Downregulation of Expansin 3 Is Not Associated with a Visible
Phenotype |
Cotton
fibers and seed oil are of tremendous economic importance. Attempts to improve cotton through genetic manipulation
have been largely successful, but there are still many traits that need to be
optimized to reduce inputs and improve fiber quality. The complex nature of the allotetraploid
genome, as well as novel characteristics of the cotton seed itself have proven
to be a challenge for finding and characterizing important genes. Expansins are a family of cell wall proteins
in plants that are known to assist in cell elongation. There are at least 6 members of the expansin
gene family in cotton, and two members are specifically expressed in fiber. The
expansins are therefore expected to be a good target for improving
fiber-related traits in cotton.
Virus-induced gene silencing (VIGS) vectors are an established means of
quickly understanding gene function in plants by reverse genetics. We used the geminivirus Cotton Leaf Crumple
Virus (CLCrV) to silence two expansin genes specific to vegetative (Expansin3) and fiber tissues (Expansin1) respectively. The object of both was to determine the
phenotype associated with down-regulating an expansin gene and to show that the
VIGS system could be used to silence genes in newly-developed cotton
fibers. Although RT-PCR confirmed
reduced Expansin3 expression in
vegetative tissues due to VIGS, no visible phenotype has been observed. We are still waiting for plants to flower to
analyze VIGS of Expansin1 in
fiber.
Rajewski, Alex C. |
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Home
Institution: |
Drake University |
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Program: |
Synthetic
Biology Research Experience for Undergraduates |
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College: |
CALS |
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Department(s): |
Molecular and Structural Biochemistry |
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Research
Mentor(s): |
Linda Hanley-Bowdoin/Molecular and StructuralBiochemistry Mary Beth Dallas/Molecular and Structural
Biochemistry |
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Title of
Presentation: |
Comparison of the symptoms caused by four geminiviruses in a common host |
The Geminiviridae are a family of
single-stranded DNA plant viruses that cause severe crop destruction especially
in the tropics. Much of the research
into geminivirus replication and resistance is conducted in model organisms
such as Nicotiana benthamiana, which
is not the natural host of the viruses.
Model plants are used because of their ease of cultivation and shorter
generation times. Typically, begomoviruses have narrow host ranges and cannot
be compared directly. However, unlike many plant species, N. benthamiana is susceptible to a variety of begomoviruses and can
be used to compare them in a common host. In this study, we compare the disease
properties of four begomoviruses whose natural hosts include tomato, cabbage
and cassava, which represent three families – the Solananceae, Brassicaceae and
Euphorbiaceae. These studies examine the Tomato golden mosaic virus (TGMV),
Cabbage leaf curl virus (CaLCuV), African cassava mosaic virus-Cameroon
(ACMV-Cam) and East African cassava mosaic virus-Uganda (EACMV-UG3). TGMV is a
model virus and is not associated with crop losses. CaLCuV infects cabbage,
cauliflower and broccoli fields in the U.S. The two cassava-infecting viruses
are part of a disease complex that causes Cassava Mosaic Disease (CMD) in
Africa. CMD is pandemic across Africa, resulting insufficient food supplies. We
infected N. benthamiana with ACMV-Cam
and EACMV-UG3 by bombardment of cloned viral sequences. TGMV and CaLCuV were
inoculated using Agrobacteria carrying
viral genomic DNA in T-DNA plasmids. The infected plants were monitored for symptoms and viral
DNA accumulation. These studies showed
that CaLCuV and ACMV-Cam caused the most severe symptoms on N. benthamiana. TGMV and EACMV-UG3
symptoms were significantly milder.
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Last modified June 2009 by Sharon E. Hunt