Nicole Smoot
• Biofuels are made by
converting biomass into usable
fuels (biodiesel and ethanol)
• Starches and sugars are
fermented into bioethanol
• Currently, we mix 10% ethanol
and 90% fossil fuels
• Although growing crops
specifically for biofuels is an
option, it uses a lot of land and
doesn’t reduce green house gas
emissions
• Instead, we can use agricultural
or municipal waste
• Second generation biofuel (manufactured from biomass)
• Made by converting cellulose into ethanol
• Current procedure:
• 1) Pretreatment to separate cellulose from lignin
• Lignin is a long polysaccharide that prevents the breakdown and
fermentation of cellulose.
• 2) Cellulolytic Process breaks down cellulose
• Cellulose is broken down either with chemicals or enzymes
• 3) Fermentation of the two resulting sugars, xylose and arabinose
• 4) Distillation
• 5) Dehydrating
• 6) Denaturing
• Corn is one of the most abundant crops grown in US (32% of
the world’s corn), so there is a lot of potenial biofuel raw
material.
• Already, most corn is genetically modified
• Corn stover: leaves and stalks of maize. Usually, stover is left in
the fields after harvest.
• Corn stover is high in cellulose, but also contains about 20%
lignin
• Currently it is expensive and inefficient to break down lignin.
• Designed to protect the plant from being eaten by bacteria, which makes
it hard to degrade
• Amycolatopsis is a soil
bacteria found in Idaho
• It is able to break down lignin
• Produces a catalaseperoxidase enzyme called
Amyco1that depolymerizes
lignin
• Capable of surviving with only
lignin as a source of carbon
• Specific strain to be used:
Amycolatopsis sp. ATCC
39116 strain 75iv2
• An intein is a section of amino acids in a protein that can selfsplice out
• Inteins splice out at random times
• Mutagenic PCR intentionally causes mutations in a gene
(approximately 2% will have a mutation)
• A collection of slightly mutanized versions of a gene is called a
library.
• By doing mutagenic PCR enough times on the intein, an intein
might be created that is induced to splice by cold.
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Corn
Cold
Lgingin
What is library
What is intein
Not out of dna out of protein
Way of testing if intein is functonslal
Why is rfp
Agro get in how not liq rub wound
What mpcr
Da f is da FLAG for?
1) Create an intein library
and insert into GFP gene.
Put into Agrobacterium
tumefaciens and cultivate on
plates.
2) Isolate colonies that begin
to glow when exposed to
cold.
3) Cut out intein nucleotide
sequence from plasmid and put
into a gene for Amyco1 in a
new vector with RFP. Insert new
4) Cultivate Agro cells in a vector into new Agro cells.
liquid buffer with corn stalk.
Test to see if freezing the
culture results in decreased
lignin.
5) Inoculate maize plants
with Agro. Test to see if the
Amyco1 enzyme is functional
only when the plant is frozen
• Perform mutagenic PCR on the intein (Clontech) and insert into a
GFP (green fluorescent protein) gene (LifeTechnologies).
• Insert the GFP gene into a Ti plasmid (tumor-promoting and
opine-synthesis genes removed) from Agrobacterium
tumefaciens (Clontech).
• Replace the plasmid inside the Agrobacterium tumefaciens and
culture in broth for one day.
• Plate the Agrobacterium. Incubate at 20°C for three days.
• Check colonies under black light to see if any fluoresce.
• Let plates sit at 0°C for six hours. Return to 20°C and let sit for
12 hours.
• Check plates under black light again to see if any have started
to fluoresce.
• Isolate any candidate colonies and culture in liquid broth (one
tube per colony) for two days.
• Plate ten plates per tube. Place at different temperatures (0°C,
2°C… 20°C) for six hours. Check each plate under black light
before and after.
Variable
Control
GFP gene with mutanized
intein in Agro
GFP gene with normal
intein in Agro
GFP without intein in Agro
Agro with candidate intein Agro with candidate intein
in GFP at 0°C, 2°C… 18°C in GFP at 20°C
Agro with no intein in GFP
at 0°C, 2°C…20°C
• Cut out intein using the same restriction enzymes used when
creating the GFP gene.
• Use restriction enzymes to place chosen intein gene sequence in
the gene for peroxidase Amyco1 (ATCC). Insert gene for
Amyco1 into a Ti plasmid (Clontech), using 35S promoter. Add
the cell wall targeting signal sequence of barley alpha amylase
to the 5’ end of the Amyco1 gene (Megazyme).
• The targeting signal sequence tells the cell to send the Amyco1
enzyme to the cell wall.
• Attach a FLAG epitope to the Amyco1 gene for future
Westerns.
• On a separate promoter (TK promoter), insert the gene for RFP
(LifeTechnologies) to check if the plant was inoculated
effectively.
• Insert the plasmid into new Agrobacterium tumefaciens cells.
• Culture cells on plates, then transfer colonies to liquid broth.
• Test concentration of lignin in a piece of corn leaf using UVspectrophotometric analysis.
• UV spectrophotometric analysis is done by shining a laser into a
substance. Based on how the light is absorbed, the size and
quantity of molecules in the solution can be defined.
• Place a piece corn leaf in a tube and add transformed
Agrobacterium.
• Incubate at 20°C for a day, then freeze for six hours. Return to
20°C and incubate for a week.
• Test lignin concentration of corn leaf again using UVspectrophotometric analysis.
Test
Expected Outcome
Corn leaf (with Agro), 0°C
Decreased Lignin
Corn leaf (No Agro), 0°C
and 20°C
Normal Lignin
Corn leaf (with Agro), 20°C Normal Lignin
• Transform immature zygotic maize embryos using
Agrobacterium tumefaciens.
• Use RFP reporter gene to ensure the plants were effectively
inoculated.
• Grow embryos for 10-12 weeks.
• Run a Western blot test on normal leaves as well as leaves that
were frozen for 4 to 6 hours then returned to 20°C for 12
hours. Use anti-FLAG antibodies (Sigma-Aldrich) to identify the
Amyco1 enzyme.
• Do UV-spectrophotometric analysis of both normal leaves and
frozen and thawed leaves to test lignin concentration.
Test
Frozen corn leaves (with
Agro)
Frozen corn leaves (no
Agro)
Normal corn leaves (no
Agro)
Normal corn leaves (Agro)
Expected Outcome
Lower lignin concentration
Normal lignin
Normal lignin
Normal lignin
• Possibly no cold-regulated inteins produced
• Cold-regulated intein may only work in GFP
• DDUN FORGET DIS LKGJLKSD diff btwn cold n chance may
confuse bc we dk if it is just doing da splicing randomly or
actually bc of cold yo
• Amyco1 enzyme might not be functional in maize
• The cell wall targeting sequence may not attack the cell wall in
maize
• Could make cellulosic ethanol as inexpensive as corn ethanol
without competing with food for land
• This process could be transferred to other plants that can grow
in marginal land (ex. some grass species)
• Biofuels could become more widespread and decrease our
dependence on fossil fuels
• Special thanks to all our instructors, Dr. Paul Feldstein, Dr. LeAnn
Lindsay, Dr. Adam Telleen, Ann Moriarty, and Dana de Farcy
for answering all of my questions.
• Thanks to all the authors I cited in this presentation.
• Thanks to the Cluster 1 fam, you guys are the best.
• Bugg, Timothy et al. "The Emerging Role for Bacteria in Lignin Degradation and Bioproduct Formation." The Emerging Role for Bacteria in Lignin Degradation and Bio-product
Formation. ScienceDirect, 9 Nov. 2010. Web. 29 July 2015.
http://www.sciencedirect.com/science/article/pii/S0958166910001977
• Shen, Binzhang et al. "Engineering a Thermoregulated Intein-modified Xylanase into
Maize for Consolidated Lignocellulosic Biomass Processing." Nature Biotechnology, 31
May 2012. Web. 29 July 2015.
http://www.nature.com/nbt/journal/v30/n11/full/nbt.2402.html#methods
• Frame, B et al. “Genetic Transformation using maize immature zygotic embryos." National
Center for Biotechnology Information. U.S. National Library of Medicine, 2011. Web. 29
July 2015. http://www.ncbi.nlm.nih.gov/pubmed/21207278
• Das, A., and S. Stachel. "Promoters of Agrobacterium Tumefaciens Ti-plasmid Virulence
Genes." Nucleic Acids Research. US Library of Medicine, 1986. Web. 29 July 2015.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC339509/pdf/nar00272-0225.pdf
• Zahm, P., SL Rhim, and K. Geider. "Promoter Activity and Expression of Sequences from Tiplasmid Stably Maintained in Mammalian Cells."National Center for Biotechnology
Information. U.S. National Library of Medicine, 1989. Web. 29 July 2015.
<http://www.ncbi.nlm.nih.gov/pubmed/2481809>
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of_UV-_visible_spectrophotometer.png
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176
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