Cloning and expression in Saccharomyces cerevisiae of the NAD(P)Hdependent xylose reductase- encoding gene (XYLI) from the xyloseassimilating yeast Pichia stifltis
Bianca Peixoto Correia, November 25, 2014
Due to increasing population and industrial development, the market for machines as well
as the automotive market is growing accordingly to meet current economic demands. Currently,
the production of ethanol is predominantly based on the use of sugar cane and the conversion of
starch derived from grain, especially corn. In recent decades because of the growing concern
about global warming, sustainability and ethics, high investment has been applied in the
development of alternative fuel sources, and the utilization of yeasts for the fermentation process
of biomass and residual biomass has presented important role.
Lignocellulosic biomass employed in the fermentation process is composed mainly of
cellulose, hemicellulose and lignin, being xylose and cellulose the most abundant sugars in
cellulose hydrolysis. Among the organisms used in industrial fermentation processes for
producing ethanol, the yeast Saccharomyces cerevisiae is the better known and through an
anaerobic process is capable of converting glucose to ethanol. Although this yeast presents the
basic mechanism of xylose assimilation, this organism is not able to utilize xylose as a carbon
source for the production of ethanol. Because of this inability of S. cerevisiae, efforts have been
made in order to make this competent yeast for fermentation of xylose.
Among the approaches used for the genetic modification of S. cerevisiae targeting the
metabolic pathway of xylose, the main are based on modeling, flux analysis, and deletion and /
or alteration in the expression of key genes. In this study conducted by Amore at al. The XYLI
gene of the yeast Pichia stipitis encoding a key enzyme in the pathway for xylose was cloned
and expressed in Saccharomyces cerevisiae in order to make this organism capable to conduct
the fermentation of xylose to ethanol.
This research represents a considerable industrial economic advantage, since it aims at
the utilization of a major of part of the waste biomass that before could not be used in
fermentation processes. Additionally, this technology opens scope for the use of alternative
carbon sources, providing an environmentally sustainable technology.
1.
2.
3.
4.
References:
Amore Rene, Kotter Peter, Kiister Christina, Michael Ciriacy, Hoilenber P. Cornelis.
1991. Cloning and expression in Saccharomyces cerevisiae of the NAD(P)H-dependent
xylose reductase- encoding gene (XYLI) from the xylose-assimilating yeast Pichia
stifltis. Gene, 109 (1991) 89-97.
Kim Soo rin, Park Yong-cheo, Jin Yong-su, Seo Jin-ho. 2013. Strain engineering of
Saccharomyces cerevisiae for enhanced xylose metabolism. Biotechnology Advances, 31:
851-856
Thomas W Jeffries. 2006. Engineering yeasts for xylose metabolism. Current Opinion in
Biotechnology, 17:320-326.
Watanabe Seiya, Saleh Ahmed Abu, Pack Pil Seung, Annaluru, Kodaki Tsutomu,
Makino Keisuke. 2007. Ethanol production from xylose by recombinant Saccharomyces
cerevisiae expressing protein engineered NADP+-dependent xylitol dehydrogenase.
Journal of Biotechnology, 130: 316-319
1
Abstract Received Late
The Flavr Savr Tomato
Lu Tian, November 25, 2014
Abstract
The Flavr Savr tomato was introduced as the first genetically engineered
whole food in 1994. The commercial event, resulting from transformation
with an antisense expression cassette of polygalacturonase gene.
Polygalacturonase is expressed in tomato only during the ripening stage of
fruit development. PG becomes abundant during ripening and has a major
role in cell wall degradation and fruit softening. Tomato plants were
transformed to produce antisense RNA from a gene construct containing
the cauliflower mosaic virus 35S promoter and a full-length PG cDNA in
reverse orientation. The construct was integrated into the tomato genome
by Agrobacterium-mediated transformation. The constitutive synthesis of
PG antisense RNA in transgenic plants resulted in a substantial reduction
in the levels of PG mRNA and enzymatic activity in ripening fruit. However,
after the gene was sequenced, people found that it contain two contiguous,
linked, transfer DNA insertions. Also, they found polygalacturonase
suppression correlates with accumulation of ’21-nt small interfering RNAs,
the hallmark of an RNA interference-mediated suppression mechanism.
Sources used:
Sheehy RE, Kramer M, Hiatt WR. Reduction of polygalacturonase activity
in tomato fruit by antisense RNA. Proc Natl Acad Sci U S A. 1988
Dec;85(23):8805–8809.
Krieger EK, Allen E, Gilbertson LA, Roberts JK. The Flavr Savr tomato, an
early example of RNAi technology. HortScience 2008; 43: 962–964.
2
RNAi can identify new components of the p53 pathway &
Silence mutant p53 pathway
By Chelsey Maag
November 25, 2014
Using experimental therapy to learn more about molecular carcinogenesis using the
powerful tool of RNAi, RNA interference, to screen for loss-of-function genes and identifying
components of cellular signaling pathways and its affect on tumourgenesis. I focus on
identifying new components of the p53 suppression pathway. Additionally, I focus on using
RNAi therapeutics on mutant p53 that accumulates typically in cancer cells. RNAi is a defense
mechanism triggered by double-stranded RNAs, which processed into short interference RNAs,
siRNAs. They target homologous RNAs for destruction. Searching for components of the p53
tumor-suppressor pathway. P53 tumor suppressor stops the formation of tumors the p53 protein
binds to DNA that stimulates another gene to produce a protein called p21 that interacts with the
next stage in cell division.
Mutations in the p53 gene, encoding the p53 tumor suppressor, are possibly the most
common type of gene specific changes in human cancer. This emphasizes the essential role of
p53 as a major support of the body’s built in anticancer defense mechanisms. P53 mutants
possess the characteristics of oncogenes, suggesting that knockdown of mutant p53 may restrict
or reverse the process of oncogenesis. Therapeutics based on RNA interference RNAi has
become powerful and useful methods for the treatment of many diseases, including cancer; due
to the high specificity, high efficacy and low toxicity of the RNAi trigger, small dsRNA. siRNA
targeting mutant p53 could induce cell cycle arrest and apoptosis in human cancer cells
RNA interference (RNAi) is a powerful new tool with which to perform loss-of-function
genetic screens in lower organisms and can greatly facilitate the identification of components of
cellular signaling pathways and stoppage of mutant p53 constant cellular proliferation. A
potentially useful application of short interfering siRNA screening is in the identification of manmade lethal interactions, which are a combination of two non-lethal mutations, that together
result in cell death. The identification of such genetic interactions in mammalian cells may
accelerate the development of new and more specific classes of anticancer drugs.
References:
Berns, Katrien, and Roderick L. Beijersbergen. "A Large-scale RNAi Screen in Human Cells
Identifies New Components of the P53 Pathway." Nature.com. Nature Publishing Group,
25 Mar. 2004. Web. 16 Nov. 2014.
<http://www.nature.com/nature/journal/v428/n6981/full/nature02371.html>
Bin Zhu,, Hai-, and Kai Yang. "Silencing of Mutant P53 by SiRNA Induces Cell Cycle Arrest
and Apoptosis in Human Bladder Cancer Cells." WJSO. World Journal of Surgical
Oncology, 28 Jan. 2013. Web. 16 Nov. 2014. http://www.wjso.com/content/11/1/22
3
Genetically Modified Cotton: To Bt or not to Bt?
Bethany Zumwalde, November 25, 2014
Cotton is one of the most important cash crops in the world accounting for over $25
billion of revenue annually in the United States (USDA 2014). Monsanto first genetically
modified cotton in 1996 to express insecticidal genes derived from the soil bacterium Bacillus
thuringiensis. This bacterium naturally produces toxic crystalline proteins (Cry-proteins) that
target specific pests such as the bollworm. When the insect larvae ingest the plant, the Bt protein
is then activated by the high pH of the gut and subsequently attaches to a specific receptor found
only in lepidopteron species (Ostlie et al. 1997). The protein then dissolves the gut lining of the
insect, ultimately resulting in death.
Bt proteins have proven to be a powerful insecticidal tool for cotton due to its high
specificity for certain insect species, non-toxicity to non-target species, biodegradability,
increase in crop yield, and also the reduction of the use of other possibly harmful insecticides.
However, the cost of the transgenic seeds can be relatively high and unaffordable for some
farmers and contemporary studies have revealed that the first generation of transgenic Bt cotton
has been proven to be susceptible to insect resistance (Tabashnik et al. 2008). A recent study in
2012 conducted by Ghazanfar et al. analyzes the inheritance of the insecticidal gene cry1Ab in
genetically modified cotton. This study incorporates two transgenic lines (CEMB-3 and CEMB11) and two non-transgenic lines (MNH-93 and CIM-482) to decrease recent field-evolved insect
resistance to first generation Bt cotton. Results from PCR, southern blots, western blots, and lab
biotoxicity assays were used to analyze and confirm high heritability of the cry1Ab gene in
addition to other cry-genes for future breeding programs to combat insect resistance.
References
1. Ghazanfar, M., Hhan, G. A., Bakhsh, A., Riazuddin, S., & Husnain, T. 2012. Inheritance of
an insecticidal gene (cry1Ab) in genetically modified cotton. Russian Agricultural Sciences,
38(3), 210-217.
2. Ostlie, K.R., W.D. Hutchison, and R.L. Hellmich. 1997. Bt corn and European corn borer.
NCR Publication 602. University of Minnesota, St. Paul.
3. Tabashnik, B. E., Gassmann, A. J., Crowder, D. W., & Carrière, Y. 2008. Insect resistance to
Bt crops: evidence versus theory. Nature biotechnology. 26(2), 199-202.
4. United States Department of Agriculture (USDA). 2014. Cotton and Wool. Retrieved from
http://www.ers.usda.gov/topics/crops/cotton-wool.aspx.
4
Roundup Ready corn: A case study
Harlan Svoboda, 25 November 2014
Roundup Ready corn, a genetically modified (GM) strain of Zea mays designed to be
resistant to the herbicide Roundup, has been in use in the United States since 1998 (Roundup
Ready System, accessed 18 November 2014). The effectiveness of this system has been proven
repeatedly (The Monsanto Company, 2002), but the toxicity of ingesting the GM food, although
proven safe, has still been in question by the public and some sects of the scientific community.
The first major criticism to the Roundup Ready corn system reported a myriad of health
problems seen in lab rats (Séralini et al., 2012). Among many disturbing accusations, the 2-yearlong study claimed that 1) all treated groups died 2-3 times more than controls, and more rapidly;
2) females produced large mammary tumors more often; 3) pituitary glands were the second
most disabled organ; 4) sex hormonal balance was modified; 5) liver congestions and necrosis
were 2.5-5.5 times higher; 6) kidney nephropathies were also generally 1.3-2.3 greater; and 7)
76% of the altered parameters were kidney related. Not only did the experimenters feed Roundup
Ready corn to the rats, comprising 11% of the rats’ diet, but also added in concentrated Roundup
herbicide into their water. The paper concluded that both Roundup herbicide and Roundup
Ready corn were toxic to rats and implied the same for human consumption.
Almost immediately, the Séralini et al. (2012) study came under scrutiny from the
scientific community. Butler (2012) articulated these concerns in a Nature article, reporting that
“the design, reporting, and analysis of the study are inadequate,” and that the paper is “of
insufficient scientific quality to be considered as valid for risk assessment.” Nearly a year after
the initial study, Arjó et al. (2013) summarized the discourse within the scientific community
regarding the poor science behind Séralini’s article, asserting that the study was designed poorly,
unsubstantiated statements were made, control data was not presented, and that the rats were
treated inhumanely. Ultimately, Arjó et al. (2013) and others insisted that the original Séralini et
al. (2012) article be retracted by the publishing journal, which it later was.
References
2014. Roundup Ready System. The Monsanto Company (accessed online at
http://www.monsanto.com/weedmanagement/pages/roundup-ready-system.aspx).
Arjó, G., M. Portero, C. Piñol, J. Viñas, X. Matias-Guiu, T. Capell, A. Bartholomaeus, W.
Parrott, & P. Christou. 2013. Plurality of opinion, scientific discourse and pseudoscience:
an in depth analysis of the Séralini et al. study claiming that RoundupTM Ready corn or
the herbicide RoundupTM cause cancer in rats. Transgenic Research 22: 255–267.
Butler, D. 2012. Hyped GM maize study faces growing scrutiny. Nature 490: 158.
The Monsanto Company. 2002. Safety Assessment of Roundup Ready Corn Event GA21
(retrieved from http://www.monsanto.com/products/pages/product-safetysummaries.aspx).
Séralini, G.-E., E. Clair, R. Mesnage, S. Gress, N. Defarge, M. Malatesta, D. Hennequin, J.
Spiroux de Vendômois. 2012. Long term toxicity of a Roundup herbicide and a
Roundup-tolerant genetically modified maize. Food and Chemical Toxicology 50: 4221–
4231.
5
Abstract Received Late
Photobiological Hydrogen Production Using
Bioengineered Algae and Cyanobacteria
Curtis Moore, November 25, 2014
No matter ones stance on climate change it is obvious that emissions from ICEs (internal
combustion engines) are no conducive to a healthy environment, any naysayer can feel free to
huff a tailpipe. On the other hand, if one were to huff a tailpipe of a fuel-cell vehicle powered by
clean hydrogen, they would be taking in only heat and water.
As of yet there are few feasible methods to produce clean hydrogen without the addition of nonrenewable energies to purify the gas. One method of production being considered by scientists
is the use of Algae and cyanobacteria to produce this gas for us. Hydrogenase enzymes are
used as a catalyst by these organisms to fix Co2 into carbohydrates using ATP and NADPH. In
the absence of CO2 and under anaerobic conditions, reduced ferrodoxin, the main electron
donor to NADPH, reduces protons to yield hydrogen gas.
Though promising, there are still challenges to using this technology to produce hydrogen on a
commercial scale. Specifically, for algae light conversion efficiency o H2 is theoretically about
10%, and the Fe-Fe hydrogenases are inhibited by oxygen produced in one of their
photosystems. These limitations are overcome by truncating the chlorophyll antenna size of PSII using RNAi and Sulfur deprivation.1
Another main limitation of this technology is competition between hydrogenases and the
NADPH-dependent carbon dioxide fixation.2 Recent experiments have shown that
bioengineering a ferredoxin-hydrognease fusion enzyme can switch the bias of electron transfer
from FNR, to hydrogenase and results in an increased rate of hydrogen photoproduction.2
In response to the experiments described above, it was concluded that a certain level of FNR
activity is necessary for stable photosynthetic growth. In response another group of researchers
genetically modified residues of PETF (photosynthetic electron transport ferredoxin) which are
essential for differential recognition of FNR and HYDA1.3
Sources used:
Singh, Shailendra Kumar.; Dixit, Kritika.; Sundaram, Shanthy. Bioengineering of Biochemical
Pathways for Enhanced Photobiological Hydrogen Production in Algae and Cyanobacteria. Int.
J. Biotech. Bioen. Re. [Online] 2013 511-518
Yacobya, Iftach.; Pochekailova, Sergii.; Toporikb, Hila.; Ghirardic, Maria L.; Kingc, Paul W.;
Zhang, Shuguang. Photosynthetic electron partitioning between [FeFe]-hydrogenase and
ferredoxin:NADP+-oxidoreductase (FNR) enzymes in vitro J Biol Chem 2013 288 (49) 3519235209
Rumpel, Sigrun.; Siebel, Judith F.; Farès, Christophe.; Duan, Jifu.; Reijerse, Edward.;
Happe, Thomas.; Lubitz, Wolfgang.; Winkler, Martin. Enhancing hydrogen production of
microalgae by redirecting electrons from photosystem I to hydrogenase R. Soc. Chem. 2014
3296-3301
6
Genetically engineering bean golden mosaic virus resistance in the common bean
Michael Xie—December 2, 2014
Bean golden mosaic virus (BGMV) is a plant virus that infects the common bean plant
(Phaseolus vulgaris L.). The whitefly (Bemisia tabaci) is virus’ insect vector. Those infected will
demonstrate the following symptoms; stunted growth, yellow-green mosaic pattern of leaves,
and distorted pods. BGMV is currently a large contributor to the annual yield loss of bean
production in Latin America. It has been estimated that the annual yield losses are 90,000280,000 tons in Brazil due to BGMV. Currently no bean plant has developed or exhibited a
natural immunity to this virus.
In order to combat BGMV, a transgenic bean line has been engineered by the company
Embrapa to be resistant by using RNA interference. The gene most important for viral
replication is the AC1 gene in DNA-A, which encodes for the complex, multifunctional
transcription factor needed for replication. The linear vector, pBGMVRNAiAHAS, was
engineered to encode RNA sequence specifically corresponding to AC1 in the sense and antisense orientation in order to elicit RNA interference. When AC1 is present in the plant cell, a
double stranded RNA complex will form and be cleaved by a Dicer (RNase III), into siRNA that
will bind to target mRNA that will be degraded by the RISC protein complex. The transgene was
introduced into the common bean genome through micro-projectile bombardment. Several lines
of transgenic beans were created and screened by virus inoculation and one was found to have
the highest expression of resistance rest (5.1). There was no viral DNA found in 5.1 when
screened through PCR with primers flanking BGMV. When 5.1 were allowed to set seed as the
founder line, Southern blot analysis revealed that the transgene were not independently
segregating.
Utilizing RNAi in plants is a novel method that has been approved by Brazil’s Nation
Technical Commission for Biosecurity. Thus, it has been approved to be substantially equivalent
and safe to its nontransgenic counterpart. Controversy surrounds safety, affecting biodiversity,
and creating an unfair advantage over smaller scale farmers.
References:
Aragão, F., Nogueira, E., Tinoco, M., & Faria, J. (2013). Molecular characterization of the first commercial
transgenic common bean immune to the Bean golden mosaic virus. Journal of Biotechnology, 166, 42-50.
Bonfim, K., Faria, J., Nogueira, E., Aragao, F., & Mendes, E. (2007). RNAi-Mediated Resistance to Bean golden
mosaic virus in Genetically Engineered Common Bean. The American Phytopathological Society,
6(10.1094), 717-726. Retrieved November 26, 2014, from
http://apsjournals.apsnet.org/doi/pdf/10.1094/MPMI-20-6-0717
CTNBio. (2010, January 1). Technical Opinion No. 3024/2011 - Commercial Release of genetically modified bean
resistant to Bean Golden Mosaic Virus (Bean golden mosaic virus - BGMV), event Embrapa 5.1 - Case
No. 01200.005161/2010-86. Retrieved November 26, 2014, from
http://www.ctnbio.gov.br/index.php/content/view/17813.html
7
No Abstract Received
Owen Riemer, December 2, 2014
Abstract
Not received
8
No Abstract Received and No Topic Chosen
Colin Hinton, December 2, 2014
Abstract
Not received
9
Abstract Received Late
Targeting and killing of malignant gliomas with specific stem cells expressing
the suicide gene, thymidine kinase, from the herpes simplex virus
Ethan Whipp, December 2, 2014
In today’s society, despite continual advances in neurosurgery and advanced radio- and
chemotherapy, a poor prognosis is often given when diagnosed with malignant gliomas. A
glioma is a tumor that arises from glial cells in the brain or spinal cord, and is responsible for
80% of malignant brain tumors (Goodenberger, Jenkins 2012). The survival rates are low, a
reported 30% of diagnosed patients live one year and only 14% of patients make it to two years
(Rachet et. al. 2008). The low survival rates are attributed to the ability of a single tumor cell,
from a glioma, to migrate to a distant area and affect healthy brain tissue, forming new tumors.
Adult stem cells can be used to target these tumors, and have the ability to target mobile glioma
cells that metastasize to healthy CNS tissues. Cultivating these adult stem cells on a large scale is
limited by low population doublings, and by the inability to subculture these stem cells
effectively. Clinical use of adult stem cells would therefore be impractical at this time, due to
these limitations. In this study, a promising strategy was developed to treat gliomas with tumor
infiltrative adult stem cells that express therapeutic genes. The adult stem cells were selected to
be highly proliferative, able to migrate to and infiltrate tumor cells, and able to be subcultured in
order to maintain cell lines. The stem cells used in this experiment were a subcategory of
mesenchymal stem cells named bone marrow-derived tumor-infiltrating cells (BM-TICs). The
BM-TICs were transduced by the retro-viral vector expressing the thymidine kinase gene from
the herpes simplex virus as well as the green fluorescent protein gene variant (BM-TIC-tk-GFP).
BM-TIC-tk-GFPs were injected into or nearby malignant gliomas in a rat model. Gene
expression was monitored via positron emission tomography using a specific tracer to confirm
expression of thymidine kinase. Fluorescence microscopy of histological sections confirmed
GFP containing cells location around the gliomas as well as infiltrating into the tumors. Rats
injected with BM-TIC-tk-GFPs were treated with the prodrug ganciclovir. Ganciclovir is an
antiviral prodrug that is phosphorylated by the viral thymidine kinase to produce a highly toxic
triphosphate. The triphosphate is then incorporated into DNA, halting replication in the S phase
and in turn signaling apoptosis. Gap junctions formed between therapeutic cells and glioma cells
allow viral thymidine kinase diffusion into the tumor, thus making them susceptible to
ganciclovir. The rats with BM-TIC-tk-GFPs treated ganciclovir lived significantly longer than
control groups. The survival analysis showed a 65% long term survival and was statistically
significant (P<0.001). Therapeutic genes incorporated into tumor infiltrating cells were shown to
be highly effective in the treatment of brain tumors in rats, and shows promise for the treatment
of humans with malignant gliomas in the future.
References:
Rachet B, Mitry E, Quinn MJ, et al; Survival from brain tumours in England and Wales up to 2001. Br J
Cancer. 2008 Sep 23;99 Suppl 1:S98-101
Goodenberger ML, Jenkins RB (2012). “Genetics of adult glioma”. Cancer genet.
doi:10.1016/j.cancergen.2012.10.009
Miletic, Hrvoie, et. al. “Bystander Killing Of Malignant Glioma By Bone Marrow-Derived TumorInfiltrating Progenitor Cells Expressing A Suicide Gene.” Molecular Therapy: The Journal Of The
American Society Of Gene Therapy 15.7 (2007): 1373-1381.
10
Abstract Received Late
Transgenetic Cell Base Therapy to ALS
Tianyi Cai, December 2, 2014
In this experiment, researchers utilized encapsulated genomic modified human
mesenchymal stromal cells (hMSC) which can produce Glucagon-like peptide 1 (GLP1) to
reduce the onset of ALS because the neuroprotective potential of GLP1 in the SOD1(G93A)
transgenetic mice model that has familial ALS. Superoxide dismutase 1(SOD1) is an enzyme
that is encoded by SOD1 gene which located on human chromosome 21; and Glycine 93
changed to alanine (G93A) represents a mutation which will influence the produce level of
SOD1, this is one of the mutations causes familial ALS. GLP1 will be utilized in the experiment
is a candidate for treatment of neurodegenerative disorder. According to previous study, GLP
shows anti-oxidant capacity and neuroprotective against excitotoxicity in vitro and vivo. To
achieve the constant drug delivery, the researcher used microcapsules contain cells; the
transgenetic cells in the capsules can produce GLP constantly like an osmotic pump. However,
the cells contained capsule do not require maintains and partials replacement. Genomic modified
MSCs were immortalized by transduction with the human Telomerase Reverse Transcriptase
gene. The cells were sealed in the spherical shape alginate matrix. And then the microcapsules
were injected to the brains of mice. According to the visual comparison between the control
group and treated group, mice in the treated group had longer lives and slower disease onset. On
the other hand, the histological analysis of brain tissue of mice shows that the mice in treated
group has lower brain injury which expressed as high microtubule associated protein 2
percentage when stain. Therefore, the experiment proved that the GLP1-hMSC therapy exhibited
to control ALS onset and increase the life time of disease.
References:
Knippenberg S, Thau N, Dengler R, Brinker T, Petris; Intracerebroventricular
injection of encapsulated human mesenchymal cells producing glucagon-like peptide 1
prolong survival in a mouse model of ALS. PLoS One, 2012.
Lewis C, Suzuki M; Therapeutic applications of mesenchymal stem cells for
amyotrophic lateral sclerosis. Stem Cell Research & Therapy, 2014, 5.
Keifer O, O’Connor D, Boulis N; Gene and protein therapies utilizing VEGF for
ALS. Pharmacology & Therapeutics, 2014, 141.
11
Novel Molecular Beacon Probe-Based Real Time RT-PCR Assay for
Diagnosis of Crimean-Congo Hemorrhagic Fever Encountered in India
Sawyer Ellis- December 2, 2014
One of the most specific chemical reactions to take place in nature is the pairing of a
nucleic acid to its complement (Sanjay 1). Using this concept, molecular beacons were
developed to show when this specific binding occurs. Molecular beacons are single stranded
nucleic acids that contain two main regions. These regions are the stem and loop. The loop
section contains a DNA target sequence, and the stem is composed of two arm segments. One
arm segment contains a fluorophore, and the other arm possesses a quencher molecule. The
model fluorophore and quencher Sanjay uses are EDANS and DABCYL, respectively. Normally
the fluorophore absorbs light and emits it back off, but fluorescence resonance energy transfer
occurs and the DABCYL absorbs the emitted energy and gives it back off as heat, thus no light is
produced. When the compliment strand of DNA binds to the DNA sequence of the loop, a
conformational change occurs that causes the quencher molecule to no longer absorb the light
energy emitted by the fluorophore and an observable change in light may be observed.
Kamboj et. al. has developed a method using this premise, coined MB Real Time RTPCR in order to detect Crimean-Congo Hemorrhagic Fever (CCHF) which is an emerging
zoonotic disease. This method has been proven to be sensitive, specific, and reproducible using
these molecular beacons. The TaqMan Assay was the previously used method, but in this study,
the MB real-time RT-PCR method proves to be more sensitive, contain less error, and has a
higher efficiency.
References:
Garrison, A. R., Alakbarova, S., Kulesh, D. A., Shezmukhamedova, D., Khodjaev, S., Endy, T.
P., & Paragas, J. (2007). Development of a TaqMan®–Minor Groove Binding Protein Assay for
the Detection and Quantification of Crimean-Congo Hemorrhagic Fever Virus. The American
journal of tropical medicine and hygiene, 77(3), 514-520.
Kamboj, A., Pateriya, A. K., Mishra, A., Ranaware, P., Kulkarni, D. D., & Raut, A. A. (2014).
Novel Molecular Beacon Probe-Based Real-Time RT-PCR Assay for Diagnosis of CrimeanCongo Hemorrhagic Fever Encountered in India. BioMed research international, 2014.
Tyagi, S., & Kramer, F. R. (1996). Molecular beacons: probes that fluoresce upon
hybridization. Nature biotechnology, 14(3), 303-308.
12
Golden Rice 1 and Golden Rice 2 – Eliminating vitamin-A
deficiency in third world countries
Kameron Starr, December 4, 2014
In many third world countries rice is the main source of food. Rice has many health benefits but lacks
any precursors to vitamin-A.As a result, many people in these countries suffer from vitamin-A deficiencies.
Golden Rice may solve this problem.
Golden rice 1 was first produces by means of introducing the biosynthesis pathway of provitamin A
(β-carotene). Agrobacterium-mediated transformation of plasmid pB19hpc makes this possible. Plasmid
pB19hpc includes the sequences for a plant phytoene synthase (psy) and the sequence coding for bacterial
phytoene desaturase (crtI) from a daffodil and erwinia uredovora, respectively. This plasmid produces
lycopene from naturally produced GGPP. To the researcher’s surprise, the endosperm of the rice contained
the machinery necessary to convert this lycopene into β-carotene without the need for a second plasmid
encoding the enzyme lycopene β-cyclase. The β-carotene is what gives the rice its golden color. Although
promising, to meet the daily recommended value of vitamin-A, a two year old would have to eat 7 pounds of
rice per day, and an adult would have to eat 20 pounds a day. This was solved by Golden Rice 2. Researchers
found that the limiting factor in the production of β-carotene is the psy gene (from a daffodil in Golden Rice
1). Genes encoding psy from various organisms were tested to see which would have the greatest yield of βcarotene. The psy gene from maize produced the greatest level of β-carotene (23 times that of Golden Rice
1).
Much controversy is involved in this topic and will be described in my talk.
References:
Beyer, Peter, Salim Al-Babili, Xudong Ye, Paola Lucca, Patrick Schaub, Ralf Welsch, and Ingo Potrykus. "Golden
Rice: Introducing the Beta-Carotene Biosynthesis Pathway into Rice Endosperm by Genetic Engineering
to Defeat Vitamin A Deficiency." THE JOURNAL OF NUTRITION (2002)
Jacqueline A Paine1, Catherine A Shipton1, Sunandha Chaggar1, Rhian M Howells1, Mike J Kennedy1, Gareth
Vernon1, Susan Y Wright1, Edward Hinchliffe2, Jessica L Adams3, Aron L Silverstone3 & Rachel Drake1,
“Improving the nutritional value of Golden Rice through increased pro-vitamin A content” Nature (2005)
Smith, Jeffrey M. "Golden Rice Is the Wrong Way to Supplement Vitamin A."Genetic Roulette: The
Documented Health Risks of Genetically Engineered Foods. Fairfield, IA: Yes!, 2007. 243-44. Print.
13
Unlikely Remediator: Engineering D. radiodurans for toxic metal cleanup
Morgan Etheridge, December 4, 2014
Deinococcus radiodurans is a soil bacterium capable of withstanding large amounts of
ionizing and ultraviolet radioactivity. The bacteria is 1.5 – 3.5 micrometers in diameter,
spherical, and pigmented. The natural habitat of this particular species of bacteria is unknown,
though members of the genus have been found all over the world. D. radiodurans was
discovered when an attempt was made to completely sterilize canned food using radioactivity;
the bacteria was the only organism remaining after the attempt. Bacteria within the entire genus
Deinococcus have the ability to withstand lethal amounts of radioactivity; the bacteria are the
most DNA damage-tolerant organisms every identified. It is believed that the DNA repair system
in D. radiodurans is responsible for the ability to withstand high levels of IR.
Scientists engineered Deinococcus radiodurans to remediate toxic heavy metal waste.
Heavy metal waste was targeted due to the high levels present as a result of global nuclear
weapons production between 1945 and 1986. About one third of the reported 3,000 waste sites
containing production materials are still radioactive. The wastes include uranium, mercury, and
toluene, among others. Metal resistance genes were cloned into D. radiodurans, conferring
resistance to the most common toxic metal wastes and granting the ability to transform those
metals into less toxic and less soluble chemicals. Scientists took merA locus and inserted in into
D. radiodurans. merA encodes mercuric ion reductase, which reduces highly toxic, thiol-reactive
mercuric ion Hg (II) to Hg (0), a much less toxic and nearly inert ion. The cloned mer encoded
six proteins that confer mercury resistance functions. Four different strains were created. After
insertion into D. radiodurans strains, the strains were inoculated into liquid medium containing
Merbromin or HgCl2. The most functional strain in Merbromin and HgCl2 was the recombinative
transformation strain. The researchers also showed that it is possible to introduce several
remediating functions into a single radiodurans host by combining the TDO functions of strain
MD560 into MD737. D. radiodurans has also been a study organism for information storage in a
potential catastrophe.
References:
Brim, H., McFarlan, S.C., Fredrickson, JK., Minton, K.W., Zhai, M., Wackett, L.P., Daly, M.J.
1999. Engineering Deinococcus radiodurans for metal remediation in radioactive mixed
waste environments. Nature Biotechnology: 18. 85 – 90.
Battista, J.R. 1997. Against all odds: The survival strategies of Deinococcus radiodurans. Annual
Review of Microbiology: 51. 203 – 224.
Wong, P.C., Wong, K., Foot, H. 2003. Organic data memory: Using the DNA approach.
Communications of the ACM: 46. 95 – 98.
14
Genetic Screening for Alzheimer’s Disease
Erin Thorstensen, December 4, 2014
Alzheimer’s disease (AD) is currently the most common form of dementia in
the elderly, affecting more than five million people in just the United States.
The cause of cell death and tissue loss in the brain due to AD is not entirely
known, however scientists believe amyloid plaques and neurofibrillary
tangles are most likely responsible. Four genes, APP, PS1, PS2, and APOE,
have been determined as causative elements of AD. This presentation will
focus on the APOE or Apolipoprotein E gene, which is proposed to play a role
in amyloid β aggregation and clearance as well as intracellular signaling
through low-density lipoprotein receptor-related protein (LRP).
As we have learned, single-nucleotide polymorphisms (SNPs) are often used
as biomarkers. The APOE gene has several SNPs, which are used to explore
the genetic basis of AD. The research article being reviewed in this
presentation concentrates on identifying deleterious nonsynonymous SNPs,
which, upon mutation, result in amino acid substitution likely to cause a
change in structure and therefore function of a protein. These SNPs,
associated with APOE, could be the cause of the amyloid plaques or
neurofibrillary tangles characteristic of AD.
AD has a complex genetic background as well as non-genetic factors, which
makes research for a cure difficult. However, research in the association
between SNPs and disease risk is making progress in our understanding of
diseases such as AD to where we could be able to predict and even prevent
amyloid plaque or neurofibrillary tangle formation.
References
Alzheimer's Association. (2011). Alzheimer's Disease and the Brain.
Retrieved November 25, 2014, from
http://www.alz.org/braintour/alzheimers_changes.asp
Masoodi, T. A., Al Shammari, S. A., Al-Muammar, M. N., & Alhamdan, A. A.
(2012). Screening and Evaluation of Deleterious SNPs in APOE Gene of
Alzheimer's Disease. Neurology Research International, 1-8.
doi:10.1155/2012/480609
15
Abstract Received Late
Expression of human growth hormone in the milk of transgenic rabbits with
transgene mapped to the telomere region of chromosome 7q
Ana Carolina Amaral Coutinho, December 4, 2014
Production of highly purified proteins at large scale has been a challenge in the
biotechnology field, and to accomplish a larger production of those pharmaceuticals the
transgenic technology has been used. Growth hormone, a factor controlling growth in the
vertebrates and metabolism of mammals, is an example of pharmaceutical in which researchers
have tried to implement the transgenic technology to obtain a bigger production. The absence of
human growth hormone promotes a series of deficiencies that has been treated with Growth
Hormone Replacement Therapy, an effective treatment that has stimulated a profitable market
once this hormone is required on a strong demand, and also presents big production costs. In this
article, Lipinski et al discusses the mammary gland as an efficient bioreactor from which it is
possible to obtain a relatively large amount of purified proteins once the protein production
might occur at high levels in the milk of lactating animals. In this experiment, the recombinant
human growth hormone (rhGH) was expressed in transgenic rabbits that carry in their genome an
hGH genomic sequence which is controlled by the rat WAP promoter. This 969 bp promoter
sequence contains an essential sequence that directs the expression of rhGH only in the
mammary gland. The transgene was mapped and identified as a stable transgene that can be
transmitted for the next generations maintaining its paternal position. The presence of
recombinant human growth hormone was also verified in the milk of the transgenic rabbits
female in a range up to the level of 10 μl/ml.
Reference:
Daniel Lipinski & Joanna Zeyland & Marlena Szalata & Andrzej Plawski & Malgorzata Jarmuz
& Jacek Jura & Aleksandra Korcz & Zdzislaw Smorag &
Marek Pienkowski & Ryszard
Slomski. 2012. Expression of human growth hormone in the milk of transgenic rabbits with
transgene mapped
to the telomere region of chromosome 7q. J Appl Genetics 53 (2012) 435–
442.
16
Abstract Received Late
Production of the antimalarial drug precursor artemisinic acid in
engineered yeast
Ana Virginia Guimaraes, December 4, 2014
Abstract
Malaria is a global health problem. Globally, an estimated 3.4 billion people are at risk of malaria.
WHO estimated that 207 million cases of malaria occurred globally in 2012 (uncertainty range 135–287
million) and 627 000 deaths (uncertainty range 473 000–789 000). Disease control is hampered by
the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium
falciparum. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia
annua (family Asteraceae; commonly known as sweet wormwood), is highly effective
against multidrug- resistant Plasmodium spp., but is in short supply and unaffordable to
most malaria sufferers. Although total synthesis of artemisinin is difficult and costly, the
semi-synthesis of artemisinin from microbially sourced artemisinic acid, its immediate
precursor, could be a cost-effective, environmentally friendly, high-quality and reliable
source of artemisinin. The engineering of Saccharomyces cerevisiae to produce
artemisinic acid was done by using an engineered mevalonate pathway, amorphadiene
synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua
that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The
synthesized artemisinic acid is transported out and retained on the outside of the
engineered yeast, meaning that a simple and inexpensive purification process can be
used to obtain the desired product. Although the engineered yeast is already capable of
producing artemisinic acid at a significantly higher specific productivity than A. annua,
yield optimization and industrial scale-up will be required to raise artemisinic acid
production to a level high enough to reduce artemisinin combination therapies to
significantly below their current prices.
Abstract (modified) retrieved from: Ro, D. K. et al. Production of the antimalarial drug precursor
artemisinic acid in engineered yeast. Nature 440, 940-943 (2006) doi:10.1038/nature04640.
References
CDC and Malaria (April, 2014). Centers for Disease Control and Prevention, Center for Global Health,
Division of Parasitic Diseases and Malaria. Retrieved from: cdc.gov/malaria.
Paddon, C. & Keasling, J. Semi-synthetic artemisinin: a model for use of synthetic biology in
pharmaceutical development. Nature Reviews. 12, 355-367 (2014).
Ro, D. K. et al. Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Nature
440, 940-943 (2006).
Schmid, G. & Hofheinz, W. Total synthesis of Qinghaosu. J. Am. Chem. Soc. 105, 624–-625 (1983).
World Health Organization. World Malaria Report 2013. (WHO, 2013)
17
Cloning of Extinct and Endangered Animals
Kellie Rabley
December 4, 2014
Some of the hottest topics in science today relate to the cloning of plants and animals.
There are several different methods used to accomplish this task in animals, two of which are
DNA microinjection and embryonic stem cells. This presentation will focus on creating
transgenic animals by using embryonic stem cells, which has been successful in several different
cases. As the interest and success of cloning of animals has grown, so has the debate of the ethics
surrounding it. To give a broad overview of this topic, I chose to cover two cases that
demonstrate cloning used as a conservation tool and using it a method of de-extinction.
The first of two cases that I will explore covers the genetic rescue of the endangered wild
sheep, the Mouflon. Scientists used cumulus-oocyte complexes gathered from two deceased
Mouflon found in a field and enucleated oocytes from another sheep species, Ovis aries. The
embryo was then implanted in a domestic female sheep, where it eventually developed into clone
of one of the female Mouflon. Scientists performed a PCR amplification of nine microsatellites
to confirm that the clone was genetically identical to the Mouflon donor nuclei.
The second case study I review is one where an extinct goat subspecies, the Bucardo, was
successfully cloned. Scientists used fibroblasts derived from a cryopreserved specimen to create
the embryos and enucleated oocytes from domestic goats to create the embryo. In this case two
experiments were run to determine what stage is embryonic development was best for
implantation. There was one successful pregnancy that resulted from implanting an embryo at
the 3- to 6-cell stage of embryo development. Even though the clone died minutes within birth,
they were still able to confirm that it genetically identical to the donor cells using microsatellite
markers.
There are definite pros and cons of cloning, most of which are related to ethics. Some
benefits to cloning extinct or endangered animals include species conservation and indefinite
research opportunities. On the other hand, potential consequences of cloning revolve around the
welfare of the clone animal and the cost. Overall it can be said that were is an exciting outlook
for the cloning of extinct; however it will be met with both scientific and ethical challenges
before it becomes a staple in today’s society.
Resources:
Folch, J., M.j. Cocero, P. Chesné, J.l. Alabart, V. Domínguez, Y. Cognié, A. Roche, A.
Fernández-Árias, J.i. Martí, P. Sánchez, E. Echegoyen, J.f. Beckers, A. Sánchez
Bonastre, and X. Vignon. "First Birth of an Animal from an Extinct Subspecies (Capra
Pyrenaica Pyrenaica) by Cloning." Theriogenology 71.6 (2009): 1026-034. Web. 23 Nov.
2014.
Holt, W. V. "Wildlife Conservation and Reproductive Cloning." Reproduction 127.3
(2004): 317-24. Web. 23 Nov. 2014.
Loi, Pasqualino, et al. "Genetic Rescue Of An Endangered Mammal By Cross-Species
Nuclear Transfer Using Post-Mortem Somatic Cells." Nature Biotechnology 19.10
(2001): 962. Food Science Source. Web. 1 Dec. 2014.
18
Abstract Received Late
Engineering of E.coli for the production of P3HP
Tallyne Vasconcelos Souza Gomes, December 4, 2014
ABSTRACT
P3HP is a kind of thermoplastic that is one of the alternatives for the petrochemical derived
plastic. Its biosynthesis needs expensive compounds to happen so, in order to make the process
cheaper, scientists decided to create a recombinant strain in E.coli to produce P3HP.
Previous studies with the plasmids pwQ02 and pwQ04 generated 10.1 g/L of P3HP. To optimize
this, the scientist created 5 strategies:
1) Use only the plasmids pwQ02, pwQ04 like what was done before;
2) Use a plasmid addition system with phenylalanine and tyrosine to work in a way that only cells
able to be cloned would survive;
3) Insert the genetic information necessary for the production of P3HP directly into the
chromosome;
4) Insert part of the information directly and part using plasmids;
5) A way to improve the fourth strategy, using the plasmid addition system with phenylalanine and
tyrosine.
To determine the plasmid stability, the samples were removed from the flask and put into plates
with and without ampicillin, to see if this antibiotic has any impact on the experiment.
The results showed that ampicillin helped to generate more stable plasmids, resulting in a better
yield of P3HP. However, since plasmids carry redundant DNA, which leads to metabolic burden
and plasmid loss, the scientists decided to come up with two strategies to overcome this: the first
one used an amino acid plasmid addition system and the second one used chromosomal
integration. They found out that those strategies worked better together, and mixed them.
The best final result was the production of 25.7 g/L of P3HP in aerobic fermentation.
19