Arom-

1. Arom- ‘Clock . • Safety & Laboratory Methods • As a prerequisite for working in the CAPS Bioscience laboratories, all team members were required to read through an comprehensive safety manual that included the following topics. • General Safety • Laboratory Dress and Hygiene • Chemical Safety • Glassware and Handling of Equipment • Microbiological Safety • Our iGEM project idea was the brainchild of Brandon and Austin, who were inspired by responses to a survey of CAPS Bioscience, Business, Humans Services, and Engineering strand students of the everyday problems that they experience. After learning the basic techniques of preparing solutions, growing broth cultures, streaking plates, isolating colonies, preserving stock cultures, and autoclaving samples, work on the main project ran from late March until the end of school in May. Tim and Graham joined the project midstream, and after school was out, Graham, Jono, and Ryan worked until just prior to the jamboree. More specific methods follow: • Transformation plates were grown at 37°C overnight in an incubator. Chemically competent cells were generated using a protocol obtained from New England Biolabs (NEB) using E. coli strain HB101. Additionally, we used NEB 5-alpha Competent E. coli cells for more recalcitrant transformations.We followed the New England Biolab's High Efficiency Transformation protocol for transformation of our parts. All bacteria were cultured in LB media with either kanamycin or ampicillin, and liquid cultures for plasmid prep were grown at 37° overnight in a shaking incubator and then used. For plasmid purification from the liquid culture, an Aurum Plasmid Mini Kit (spin method) was used. Concentrations of DNA were taken using a Eppendorf Spectrophotometer at a 10ml sample to 40ml dilutant ratio. All restriction digestions were performed with New England Biolabs restriction enzymes and buffers, and the reactions were incubated for 30 minutes at 37°C followed by a 20 minute heat-inactivation step at 80°C. All gels were 2% agarose with ethidium bromide added and run at 120V until complete. A Sigma GenElute™ Gel DNA Extraction Kit was used to extract the digested DNA out of the gel. • Problem & Concept • In this busy day and age, with potential distractions, interruptions, and worries everywhere, lack of sleep is an emerging international problem. Insufficient sleep can be the cause of a host of conditions, with possible short-term effects including migraine headaches, lack of focus, poor judgment, and decreased energy and mood. Long-term effects of sleep deprivation include an increase in the likelihood of having a heart attack or stroke, and also contributes to diabetes and obesity. Sleep deprivation and bad sleep habits can arise for a number of reasons, but one of the most common is insomnia, or the inability to fall asleep when one wants to. • What is the solution to those long, sleepless nights and early, early mornings? The Arom-O’-Clock! • Our project, in essence, designs the perfect alarm clock and sleep aid. The presence of a strong wintergreen smell, produced when there is light striking the bacteria, is used to help the consumer wake up, heightening their senses. At night, the banana odor produced will assist in calming down the user, helping them fall asleep. This all occurs without the need for electricity through bacterial production of the enzymes and scents. This system is the combination of the University of Texas and MIT teams' projects from 2004 and 2006. We are making a combined genetic circuit with the smell of the bacteria (banana or mint) dependent on the time of day (presence of light). The light receptor protein, obtained from photosynthetic algae, turns the banana smell on in the absence of light, and the mint smell on in the presence of light. The broth used to culture the final strain of bacteria would need to include the substrates isoamyl alcohol and salicylic acid to produce the aromatic molecules. Austin Rottinghaus, Brandon Whitcomb, Tim Schaefer, Graham Wehmeyer, Jonathan Hermanson, and Ryan McLean Foundations & Outreach During first semester, Austin, Brandon, and Tim were introduced to synthetic biology through the BioBuidler Eau d’E. coli activity in our introductory molecular biology course. This motivated them to enroll in our research course to explore synthetic biology more fully. There they completed another BioBuilder activity, Picture This, which introduced them to the use of the modeling software Tinker Cell. In February, eight CAPS Bioscience students attended a day long Synthetic Biology workshop organized by Dr. Todd Eckdahl at Missouri Western University, and upon their return the research students attempted to complete the laboratory Using Bacterial Computers to Solve the Pancake Problem produced by Modern Biology, Inc, which was inspired by the work of the MO Western/Davidson iGEM Team. After school was out, Graham, Jonathan, and Ryan continued on the project conceived and initiated by Austin and Brandon. Our iGEM team members and other CAPS Bioscience students were involved in the following outreach: Co-transformation • Results & Future • Brandon, Austin and Tim successfully transformed the banana odor generator, mint odor generator, and the OmpC promoter. However, multiple attempts to transform the Cph8 light sensor/EnvZ complex failed. Attempts at problem solving, including using different competent cells and antibiotics, proved unsuccessful. • In June, Graham and Jono made liquid cultures of the successfully transformed parts in preparation for plasmid purification. DNA concentrations were taken with a spectrophotometer. The OmpC promoter purifications had a concentration of 25.0 ng/ml and 20.0 ng/ml and the banana odor generator had concentrations of 43.9 ng/ml and 20.0 ng/ml. Afterward, Graham and Jono performed a restriction digest on the OmpC promoter and banana smell generator in preparation for ligation, and ran a gel to separate out the cut portions of the plasmid. Graham and Jono then eluted the DNA out of the gel in preparation for ligation. • The future of the Arom-O’Clock lies in the hands of subsequent CAPS Bioscience iGEMers. Besides transforming the light sensor, which we have had difficulty with, we also need to assemble the parts on two separate plasmids, and co-transform them into competent cells that are naturally low in EnvZ. Otherwise, we are planning on collaborating with the engineering students to create a product that allows the bacteria to safely sit inside a traditional bedside clock. The Arom-O’Clock would expose the banana producing bacteria to light in order to emit the mint scent. Knowing that bacteria need pretty consistent conditions, we imagine a replaceable bacteria pack that the user could insert into the clock to refresh the bacteria supply when the odor emitted begins to fade. Catching ZZZ’s At night, when the light sensor is not active, the EnvZ protein in the membrane protein complex cph8 is more highly activated, phosphorylating a greater quantity of the transcription factor OmpR, which naturally occurs in E. coli. Large amounts of OmpR-P, in turn, will supress the OmpF promoter, which controls the mint odor generator. It will also activate the OmpC promoter, which starts transcription of the banana odor generator, the ATF1 gene, which codes for alcohol acetyltransferase (AATase) (Batchelor, et. al, 2005; Toshio, et. al., 1994). AATase catalyzes the conversion of isoamyl alcohol to isoamyl acetate which is responsible for the banana smell (Dixon & Kuldell, 2011). Rise & Shine In the daylight, when a photon hits the light sensor complex cph8, EnvZ activity is reduced, resulting in less OmpR phosphorylation. OmpR then activates the OmpF promoter, starting transcription of the mint odor generator, the BSMT1 gene. BSMT1 codes for SAM benzoic acid/salicylic acid carboxyl methyltransferase I, which catalyzes the conversion of salicylic acid to methyl salicylate, which is responsible for the wintergreen smell. Transformed Colonies OmpC Promoter Banana Generator Mint Generator • Cottonwood Point Elementary School Science outreach where 40 second graders were introduce to DNA and forensics. • CAPS Bioscience Camp where 25 middle school students completed the iconic pGLO transformation laboratory. • KSU Engineering Camp where 24 ninth graders completed the 3A Assembly Kit. • KSU Engineering Workshop where 15 middle and high school biology instructors completed a 3A Assembly paper activity created by the CAPS Bioscience instructors. • BioBrick Parts • OmpC Promoter (BBa_R0082) - A promoter we are attaching to the banana gene sequence, it activates transcription when high amounts of OmpR-P are present, which occurs when there is no light. • Banana Odor Generator (ATF1) (BBa_J45199) - The banana smell generator contains an RBS, the coding region for the yeast alcohol acetyltransferase (ATF1) ,and two terminators. ATF1 catalyzes production of isoamyl acetate, the banana smell, from its precusorisoamyl alcohol. • OmpF Promoter (BBa_K116500) - A promoter for the mint gene sequence. It is activated in the day when low amounts of OmpR-P are present. • Ribosome Binding Site (RBS) (BBa_B0030) - A ribosome binding site that will be attached to the mint odor generator. • Mint Odor Generator (BSMT1) (BBa_J45004)- Contains the coding sequence for SAM benzoic acid/salicylic acid carboxyl methyltransferase I (BSMT1). This gene was isolated from petunia and snapdragon plants. BSMT1 catalyzes the conversion of benzoic acid to methyl benzoate, which has a floral smell, and salicylic acid to methyl salicylate, which has a wintergreen smell. • Terminator 1 (BBa_B0010)- the first of the two terminators we are attaching to the mint odor gene, ends transcription. • Terminator 2 (BBa_B0012)- the last of the terminators we are attaching to the mint gene, ends transcription. • Light Sensor Protein Complex or Cph8 (BBa_I15010) - contains coding region for the light sensor Cph1 and the enzyme EnvZ. This part was taken from a UT Tech previous iGEM project. If a photon hits the light sensor Cph1 it will then inhibit EnvZ, stopping phosphorylation of OmpR. 3A Assembly Transformation Confirmation of Digest correspondence Eric Kessler & Joe Whalen Bioscience Strand Center for Advanced Professional Studies (CAPS) 7501 West 149th Terrace Overland Park, Kansas 66223 (913) 239-5900 ekessler@bluevalleyk12.org acknowledgements: Special thanks to Natalie Kuldell at BioBuilder, Todd Eckdahl at Missouri Western, and Alyssa Henning at GinkgoBioworks, who provided motivating introductory labs, a wonderful workshop, and important mentorship, respectively! references Batchelor, Eric, et. al. (2005) The Escherichia coli CpxA-CpxR Envelope Stress Response System Regulates Expression of the Porins OmpF and OmpC, Journal of Bacteriology, Vol. 187, No. 16, p. 5723-5731. James Dixon & Natalie Kuldell, BioBuilding: Using Banana-Scented Bacteria to Teach Synthetic Biology. In Christopher Voigt (2011) editor: Methods in Enzymology, Vol. 497, p. 255-271. Fujii, Toshio, et. al. (1994) Molecular Cloning, Sequence Analysis, & Expression of Yeast Alcohol Acetyltransferase Gene, Applied & Environmental Microbiology, Vol. 60, No. 8, p. 2786-2792.