HiGene 12/13 Research Project: DNA Sequence Analysis of the Duckweed Landoltia punctata

1. HiGene 12/13 Research Project: DNA Sequence Analysis of the Duckweed Landoltia punctata 

2. What is the Waksman Project? • Biotechnology and Microbiology • 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 exome and much simpler

3. Learning Aspect • cDNA libraries • Overnight Cultures • Polymerized Chain Reaction • Minipreps – Purifying the duckweed genes • Gel electrophoresis • Restriction Digest • DNA sequence Analysis

4. Labs Aspect

5. Why Join the Waksman Club? • 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 

6. Process Purify the duckweed DNA from the bacteria Grow Bacteria colony with the duckweed genes to get more DNA Do PCR and run a gel to see if the DNA is long enough and good enough Sequence the DNA Publish your sequences! Analyze the DNA

7. Duckweed: • Smallest flowering plants, • Grow in slow moving fresh water -world wide, • Fast growing - double in a few days

8. 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

9. Constructed a cDNA Library Bacteria Students will pick bacterial colonies to purify plasmid DNA containing the duckweed genes

10. 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

11. Evaluate DNA Sequence Data

12. Conduct Bioinformatic Searches of the Databases

13. Perform searches of the scientific literature to determine the function of these genes

14. Publish the DNA sequences in the public databases http://avery.rutgers.edu/WSSP/StudentScholars/WSSP10/FrameSet.html Student's and Teacher’s Names

15. RNA Interference (RNAi) Experiment • The pTRiplEx2 plasmid contains promoters on either side of the polylinker the result in the expression of dsRNA of the insert fragment. • 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. • “Bag of eggs” phenotype • The results from the RNAi experiment with Clone 20 did not produce a “bag-of eggs” phenotype, indicating the gene was not essential for embryonic development. • These results agree with RNAi studies with the C. elegans gene. 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. Sequencesanalyzed 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 genethat ishomologous to sections of the coding regions of the C. elegansptr-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 lysine-tRNA within an intron of the ptr-13 gene. This homology also occurs in other genes of C. elegans such as spe-9. Although the coding regions of ptr-13 are more highly conserved between species than non-coding regions, the size of the introns and the lysine-tRNA remains preserved. A Tale of Two Worms Julie M. Bianchini, Hillsborough High School, NJ • Structure of the C. elegansptr-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 • Introduction • Homologous genes have been conserved through evolution and often have similar functions in different organisms. • The goal of our project was to sequence regions of 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 enough for the C. remanei genes to produce the same RNAi phenotype as the C. elegans genes. • 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. • 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. • 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 . • Conclusion • Comparing the ptr-13 gene between C. remanei, C. elegans, and C. briggsae results suggest coding regions of the ptr-13 gene are more highly conserved 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. • Although genes of the patched superfamily are necessary for animal proliferation, the silencing of ptr-13 does not directly affect embryonic development. • This could result from the abundance of PTC genes that can interact with each other in order to ensure normal cell development. • Methods • A C. remanei DNA plasmid library was constructed 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. • After verification, the sequence data was submitted to the NCBI genomic database. Acknowledgements We thank the Hillsborough High School students for their research assistance, interest, and dedication to the Waksman Student Scholars Program, Rutgers University. Special thanks to: 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 Based on a figure from Alberts et al. • 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. • The wave form for a portion of the Clone 20 sequence is shown above. Present a poster on your findings

16. Timeline of the 2012/2013 HiGene Project We will work with you to fit the project into your schedule

17. We also thank the National Science Foundation and GE Healthcare for their support

18. We are grateful for the support of the Waksman Institute (Joachim Messing, Director) Dr. Selman Waksman won the Nobel Prize in 1952.

19. Goals for the Year: Learn about molecular biology and bioinformatics, have each club member isolate and publish their own sequence, and have fun!

20. 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

21. Last but not least… • Check out our website! • www.southwaksmanclub.weebly.com • Join our fb group! • Complete our participation survey (due September 26th) ~https://docs.google.com/spreadsheet/viewform?formkey=dDZsR3BPb25aZ3ZxY3h2VVRQNWwyRmc6MQ#gid=0 • Bring your dues ($5) so you can get food • T-shirt Competition (Deadline for designs Oct. 2nd)