13/14 Research Project:
DNA Sequence Analysis of the
Duckweed Landoltia punctata
What is the Waksman Project?
 Biotechnology and Molecular Biology
 Isolate and sequence duckweed’s functional
genes
 Analyze them on DSAP
 Publish sequences to the NCBI
 Similar to the human genome project but only
for the duckweed genome
 cDNA libraries
 Overnight Cultures
 Polymerized Chain Reaction
 Minipreps – Purifying the duckweed genes
 Gel electrophoresis
 Restriction Digest
 DNA sequence Analysis
 It’s a rare opportunity
 This is a real research project
 You get lab experience
 You get to be a published scientist!
 It looks great on your résumé
 We have food at every meeting
 We have fun 
Grow Bacteria
colony with
the duckweed
genes to get
more DNA
Sequence the
DNA
Do PCR and run
a gel to see if
the DNA is long
enough and
good enough
Analyze the
DNA
Purify the
duckweed
DNA from the
bacteria
Publish your
sequences!
Duckweed:
• Smallest flowering plants,
• Grow in slow moving fresh water -world wide,
• Fast growing - double in a few days
Duckweed:
• Potential biofuel source
• Under cold temperatures can accumulate 40%-70% starch
• Sink to bottom of ponds
• Starch can easily be converted to sugar for fermentation
• Will not compete with food crop production
• Bioremediation
• Grows in contaminated water
• Sequesters or degrades contaminates such as lead,
arsenate, halogenated compounds
• Extract nitrogen and phosphate from waste water
• Potential food source!
Constructed a cDNA Library
Bacteria
Students will pick bacterial
colonies to purify plasmid DNA
containing the duckweed genes
Students will determine the size of the Landoltia cDNA by
PCR and gel electrophoresis, purify the DNA and verify the
insert size by restriction digests
Evaluate DNA Sequence Data
Conduct Bioinformatic Searches of the Databases
Perform searches of the scientific literature to
determine the function of these genes
Publish the DNA sequences in
the public databases
http://avery.rutgers.edu/WSSP/StudentSch
olars/WSSP10/FrameSet.html
Student's
and
Teacher’s
Names
Present a poster on your findings
Abstract
Hillsborough High School students undertook two
projects as part of the Waksman Student Scholars
Program (WSSP), Rutgers University. One part of the
project was to sequence and analyze clones from a
genomic library made from the Caenorhabditis
remanei nematode. Sequences analyzed by high school
students in the WSSP were submitted to NCBI
GenBank. The second part of the project was to use
an RNAi method to silence gene expression in an
effort to identify genes vital to embryonic
development. We screened for C. remanei clones that
blocked embryonic development in C. elegans, a
related nematode. A C. remanei gene that is
homologous to sections of the coding regions of the C.
elegans ptr-13 gene was identified. This gene is a
member of the patched (PTC) superfamily, a class of
multipass membrane proteins that control cell
proliferation and cell fate. Interestingly, we found
this sequence from C. remanei was homologous to a
Introduction
lysine-tRNA within an
intron of the ptr-13 gene. This
homology
also occurs
in other
genes
of C. elegans
• Homologous
genes have
been
conserved
throughsuch
as evolution
spe-9. Although
thehave
coding
regions
of ptr-13
and often
similar
functions
in are
more
highly conserved
different
organisms.between species than noncoding regions, the size of the introns and the lysine• Theremains
goal of preserved.
our project was to sequence regions of
tRNA
the genome from C. remanei, a soil nematode in
which the genomic sequence has not been
determined.
• We wanted to investigate if the sequences from
this organism are strongly conserved in C. elegans, a
related nematode with a different sexual life cycle.
• We also wanted to determine if the sequences are
conserved strongly Methods
enough for the C. remanei genes
to produce the same RNAi phenotype as the C.
• A
C.
remanei
DNA
plasmid
library was constructed
elegans genes.
by partial digestion of the genomic DNA with
Sau3A and then random fragments were cloned into
a bacterial expression plasmid called pTriplEx2
(Clontech Inc.)
•
Clones from this plasmid library were purified and
the presence of the genomic inserts were verified
by PCR and restriction digestion.
•
Clones with inserts were sequenced and the
waveforms were analyzed by students at New
Jersey high schools participating in the WSSP
program.
•
RNA Interference (RNAi) Experiment
A Tale of Two Worms
Julie M. Bianchini, Hillsborough High School, NJ
• Structure of the C. elegans ptr-13 gene. Red lines show
regions of sequence similarity with the C. remanei DNA.
• Although the introns of the genes have a similar length,
only the lysine-tRNA region has been conserved through
evolution.
• After verification, sequences were deposited at
NCBI. The Accession number for Clone 20 is
CZ179566
• C. elegans strains containing sem-2 mutations
do not form a vulva and are unable to lay eggs.
As a result, the eggs hatch within the mother
and produce a “bag of worms” phenotype.
“Bag of worms” phenotype
• Transformants of clones from the C. remanei
genomic DNA library were fed to C. elegans
strain containing the sem-2 mutation.
• If the presence of the C. remanei dsRNA
blocks embryonic development through RNAi,
then the eggs will not hatch and the sem-2
worms will show a “bag of eggs” phenotype.
• Many of the other Lys-tRNAs are also coded within
intron regions of genes.
What is Ptr-13?
• Ptr-13 is a member of the Patched (PTC) related
family of genes.
Sequence Analysis
• BLASTn analysis of Clone 20 of the NCBI C. remanei
database shows strong sequence similarity to two
exon regions (green underline) of the ptr-13 gene in C.
elegans.
• Base pairs 37 to 136 ( red underline) also show strong
sequence similarity to lysine-tRNA genes from C.
elegans. Interestingly, a large number of these
genes, including a homolog of ptr-13, lie in intron
regions of genes coding for proteins .
• Ptr-13 is a multipass membrane component.
• Members of the patched superfamily function as
receptors for the hedgehog ligand and control cell
proliferation and cell fate.
• C. elegans contains at least 32 members of the PTC
superfamily which are likely involved in many
developmental processes.
• Ptr-13 is included in this family. However RNAi
experiments with this gene showed that it is not
essential for viability or development.
“Bag of eggs” phenotype
• The results from the RNAi experiment with
Conclusion
Clone 20 did not produce
a “bag-of eggs”
phenotype, indicating the gene was not
• Comparing the ptr-13 gene between C.
essential for embryonic development.
remanei, C. elegans, and C. briggsae results
codingagree
regions
of RNAi
the ptr-13
gene
are
• suggest
These results
with
studies
with
more
conserved
the C.highly
elegans
gene. between species than
non-coding regions; however, the base pair size
of introns and the lysine-tRNA remains
preserved.
• RNAi experiments using dsRNA from Clone 20
did not produce an embryonic lethal result
(bag of eggs).
• The wild type result for the RNAi agreed with
the tested genomic results.
After verification, the sequence data was
submitted to the NCBI genomic database.
Based on a figure from Alberts et al.
• The wave form for a portion of the Clone 20 sequence
is shown above.
• The pTRiplEx2 plasmid contains promoters on
either side of the polylinker the result in the
expression of dsRNA of the insert fragment.
• In the absence of Hedgehog, the Patched protein
inhibits Smoothened from signaling preventing
expression of Hedgehog target genes.
• In the presence of Hedgehog, the Patched receptor
is prevented from inhibiting Smoothened.
Smoothened is then able to signal the activation of
transcription of Hedgehog target genes.
• Although genes of the patched superfamily
are necessary for animal proliferation, the
silencing of ptr-13 does not directly affect
Acknowledgements
embryonic development.
thank the Hillsborough High School students for their research
• We
This
could result from the abundance of PTC
assistance, interest, and dedication to the Waksman Student Scholars
genesRutgers
thatUniversity.
can interact with each other in
Program,
order
to to:
ensure normal cell development.
Special
thanks
Marek Grodzicki (2003-2004 Student Scholar)
Michael Folsom-Kovarik (2004-2005 Student Scholar)
Robert Christ (2004-2005 Student Scholar)
Supervisors and Mentors:
Ms. Laura Dietz, Dr. William Sofer, Dr. Andrew Vershon, Dr. Martin
Nemeroff,
Dr. John Nelson
We thank GE HealthCare for supplying reagents and sequencing
services for the Waksman Students Scholars Program
Timeline of the 2013/2014 HiGene Project
We will work with you to fit the project into your schedule
Goals for the Year:
Learn about molecular biology and bioinformatics,
have each club member isolate and
publish their own sequence, and
have fun!
Important things to know
 We meet every Tuesday
 We split up into Freshmen and the Veterans so that
everyone can work at their own pace
 Waksman is a big commitment. If you miss meetings,
it is hard to make up what you missed.
 In addition to labs, you need time to analyze
sequences on a program called DSAP if you don’t
finish during meetings. Finch TV is easy to use on all
computers so no excuses!
 There will be deadlines for computer assignments but
there will also prizes associated with completion :D
Last but not least…
 Check out our website!
 www.southwaksmanclub.weebly.com
 Fill Out the Waksman Survey on the website
 Bring your dues ($5) so you can get food
 T-shirt Competition!
(Deadline for designs Oct. 2nd)