iGEM 2017 — Team Purdue

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:Purdue/Notebook

Toggle navigation

[![Purdue](https://static.igem.org/mediawiki/2017/d/da/Purdue_Logo.png)](https://2017.igem.org/Team%3APurdue)

* Team
  + [Team History](https://2017.igem.org/Team%3APurdue/Team)
  + [Meet the Team](https://2017.igem.org/Team%3APurdue/MeetTheTeam)
  + [Team Collaborations](https://2017.igem.org/Team%3APurdue/Collaborations)
* Project
  + [Description](https://2017.igem.org/Team%3APurdue/Description)
  + [Problem](https://2017.igem.org/Team%3APurdue/Problem)
  + [Pathway Design](https://2017.igem.org/Team%3APurdue/Design)
  + [Lung Microbiome](https://2017.igem.org/Team%3APurdue/lungmicrobiome)
  + [Genetic Construct Design](https://2017.igem.org/Team%3APurdue/GeneticDesign)
  + [Experiments and Results](https://2017.igem.org/Team%3APurdue/Experiments)
  + [Notebook](https://2017.igem.org/Team%3APurdue/Notebook)
  + [InterLab](https://2017.igem.org/Team%3APurdue/InterLab)
  + [Model](https://2017.igem.org/Team%3APurdue/Model)
  + [Demonstrate](https://2017.igem.org/Team%3APurdue/Demonstrate)
  + [Attributions](https://2017.igem.org/Team%3APurdue/Attributions)
* Parts
  + [Parts](https://2017.igem.org/Team%3APurdue/Parts)
  + [Basic Parts](https://2017.igem.org/Team%3APurdue/Basic_Part)
  + [Composite Parts](https://2017.igem.org/Team%3APurdue/Composite_Part)
* [Safety](https://2017.igem.org/Team%3APurdue/Safety)
* Human Practices
  + [Silver HP](https://2017.igem.org/Team%3APurdue/HP/Silver)
  + [Integrated and Gold](https://2017.igem.org/Team%3APurdue/HP/Gold_Integrated)
  + [Public Engagement](https://2017.igem.org/Team%3APurdue/Engagement)
* Awards
  + [Applied Design](https://2017.igem.org/Team%3APurdue/Applied_Design)
  + [Entrepreneurship](https://2017.igem.org/Team%3APurdue/Entrepreneurship)
  + [Hardware](https://2017.igem.org/Team%3APurdue/Hardware)
  + [Model](https://2017.igem.org/Team%3APurdue/Model)
* [Judging Form](https://igem.org/2017_Judging_Form?team=Purdue)

# Notebook

[Week 1](#jump1)

[Week 2](#jump2)

[Week 3](#jump3)

[Week 4](#jump4)

[Week 5](#jump5)

[Week 6](#jump6)

[Week 7](#jump7)

[Week 8](#jump8)

[Week 9](#jump9)

[Week 10](#jump10)

[Week 11](#jump11)

## Week 1

## Monday, 5/22 Day 1

**Andrew:** Work was assigned for the week. A method for identifying ribosome binding sites (RBS’s) was determined through literature review and past iGEM projects. RBS’s from 13 different lung, mouth, and throat bacteria were recorded.

**Archana:** Reassessed team budget. Distributed payroll forms to interns. Worked on Experiment Campaign and modified wiki.

**Caleigh:** Coordinated logistics of lab meetings and intern safety training and laboratory access. Delegated tasks to interns and began drafting a description of project context, significance, and goals for an Experiment fundraising campaign.

**Katie:** Performed a literature review for a method for creating a lung mimic. Found and investigated two major options: decellularization and recellularization of mammal lungs and a 3D organotypic culture using a rotating wall vessel. Attended SURF orientation.

**Kevin:** Modified the protocols for the quantification of benzene, xylene, and toluene to include the addition of appropriate levels of volatile substance. Compiled supplies list for these protocols. Began work on universal promoter.

**Vi Ka:** Reviewed recently published literature (2010-2017) for identification of different genera of bacteria living in the lung microbiome. Compiled a list to contact authors for cultures of bacteria strain. Resuspended DNA primers.

## Tuesday, 5/23 Day 2

**Andrew:** RBS’s from an additional set of bacteria were recorded for a total of 21 lung, mouth, and throat bacteria. A biography for experiment.com was written. Bloodborne pathogen training was completed. RBS’s from 18 total bacteria not found in the previous set were recorded.

**Archana:** Contacted companies regarding discounts for lab supplies. Discussed budget with Dr. Rickus. Brainstormed wiki themes and layouts. Updated wiki team page with member bios and pictures.

**Caleigh:**Finished writing the Experiment fundraising campaign proposal and constructed a GANTT chart for project deadlines.

**Katie:** Performed a literature search to create an in-vitro enzyme activity assay using the production and consumption of NADH and NAD+. Found two possible methods: using spectrophotometry and buying an NAD/NADH ratio kit. Found a variety of documents describing protocols used in past years by the iGEM team.

**Kevin:** Continued work on universal promoter by finding consensus between Pbad promoter on E. coli and other organisms.

**Vi Ka:** Continued contacting authors for bacteria culture strains. Received responses - however due to international shipping costs and customs regulations, had to turn them down.

## Wednesday, 5/24 Day 3

**Andrew:** An updated PCR protocol update and bill of materials were assigned. RBS’s from 41 total bacteria were run through comparative sequence analysis in order to compile a logo plot and to propose 5 possible “universal” sites. Through literature review, a mathematical model for transcription rates was found along with software that applies this model to given sequences.

**Archana:** Continued contacting company representatives. Updated wiki team page with member bios and pictures. Purchased supplies.

**Caleigh:** Organized agenda for today’s meeting with the advisor Dr. Rickus and the presentation for tomorrow’s meeting with the advisor with Dr. Solomon. Safety review for future project plans.

**Katie:** Prepared slides for a meeting with our PI, Dr. Rickus. Attended a literature search seminar for the Purdue Summer Undergraduate Research Fellowship (SURF) about learning vocabulary required for fully understanding the project as well as identifying papers which would be useful. Arrived late to the meeting with Dr. Rickus, but was still able to explain the two literature searches had conducted the day before. Dr. Rickus advised on some on-campus experts to contact for the lung mimicking model as well as a new method for the in-vitro enzyme activity assay using NADH fluorescence to measure its concentration. Emailed one of the team members working with decellularization whom Dr. Rickus had suggested contacting before lunch. Had a second meeting with Dr. Rickus after lunch in which SURF requirements and some other possible lung mimicking model methods were discussed. Continued the literature search and edited meeting slides with the new information that was received for the next day’s meeting with Dr. Solomon. At the end of the day, prepared LB broth and LB + agar with antibiotics for plating.

**Kevin:** Worked on logistics for the quantification of benzene, toluene, and xylene protocols. Contacted Bruce Cooper about use of Bindley’s gas chromatograph. Began research on standard procedures to determine the effects of a toxic substance on the growth of a culture.

**Vi Ka:** Received an offer from Dr. Weimer from the USDA for megasphaera elsdenii culture strain. Discussed with advisors+team regarding logistics of culturing this anaerobic bacteria. Started reviewing literature regarding transport assays of benzene/fatty acids into the cell.

## Thursday, 5/25 Day 4

**Andrew:** A better method for applying the transcription rate model to the project was described. Through literature review, papers using liquid nitrogen as a preservation method for bacteria were found as well as others using sucrose as a freezing agent. A protocol for bacterial preservation specific to the project was started keeping multiple methods and safe freezing agents in mind. The 5 chosen RBS’s for the “universal” site were uploaded for ordering. Metabolic modeling was assigned.

**Archana:** Continued contacting company representatives. Searched for best prices on lab supplies. Researched other funding opportunities.

**Caleigh:** Wrote protocols as to what gBlocks and steps are required to make specific genetic constructs. Made LB, LB-chloramphenicol, and LB-ampicillin agar plates.

**Katie:** Wrote emails to our team member and an on-campus expert, Dr. Harbin, who works with the manipulation of pig collagen, regarding the lung mimic model. Finished preparing the slides for the meeting with Dr. Solomon. Set up a House of Quality to evaluate the various methods found to mimic the lung. Continued writing the in-vitro enzyme activity assay. Had meeting with Dr. Solomon and began further research on both topics presented after receiving his advice.

**Kevin:** Continued research on substance toxicity and cell viability. Began research on how to quantify nahK protein activity. Reframed quantification of benzene, toluene, and xylene protocols on a different piece of literature. After constructive criticism from Dr. Solomon, began searching for a constitutive promoter with a common sigma factor that I could then base the universal promoter on.

**Vi Ka:** Continued literature review on transport assays - decided to use fatty acid in our protocol instead of benzene (for now), because it is harder to quantify transport of benzene into the cell. Most protocols used radiolabeled oleic acid, however due to lab regulations we won’t be able to work with radioactive materials.

## Friday, 5/26 Day 5

**Andrew:** Literature review concerning preserving bacteria was continued, exploring whether to perform this through lyophilization or freezing in harmless compounds (like sucrose). Vacuum drying was researched as another possible method. A final method was decided which included freeze-drying bacteria with sucrose and skim milk powder at various temperatures.

**Caleigh:** Attended a meeting to discuss our use of Bindley Metabolite Profiling Center equipment. Created gBlocks from the universal ribosome binding sites designed by Andrew to allow for efficient use of the gBlocks in several constructs and assays and Gibson assembly.

**Katie:** Finished the enzyme assay bill of materials for Archana. Attended a meeting with Dr. Sors and took note of other on-campus experts to contact about the enzyme assays and the lung model. Wrote the Relay for Life post for the Wiki, relating to the team’s public engagement that had been executed in the spring. Made a graphic for the Relay for Life post.

**Kevin:** Found a sigma-70 constitutive promoter for the atpI gene in E. coli that should prove effective as a basis for the universal promoter. Met with Bruce Cooper from Metabolite Processing Center about use of his gas chromatograph and received approval of benzene, toluene, and xylene quantification protocols. Decided that xylene effects poses no serious health effects and should therefore not be tested. Wiki work!

**Archana:** Continued contacting company representatives. Began intern payroll process. Continued researching corporate funding opportunities.

**Vi Ka:** Preliminary protocol for transport assay designed; will need to perform additional review.

[Back to Top](#top)

## Week 2

## Monday, 5/29: Memorial Day

Day off!

## Tuesday, 5/30 Day 6:

**Andrew:** A new protocol for preserving bacteria and testing viability was started. Tubes able to withstand overnight storage in liquid nitrogen were researched for the purpose of testing this method of preservation. The protocol was completed with room for flexibility of advice from advisor Dr. Rickus and from additional equipment. A practice run of transforming bacteria was performed to prepare for GFP assays later. Work began on metabolic pathway modeling and copying a tentative design into a Python script for personal testing.

**Archana:** Searched for lowest prices for Bill of Materials items. Contacted Ph.D. candidate, Soo, about using DTT from her lab and emailed Dr. Cooper about in-kind donations. Completed payroll process. Formulated Experiment Campaign Budget.

**Caleigh:** Ordered the Universal RBS from IDT. Began investigating United States Food and Drug Administration regulations that would affect implementation of our proposed therapy and began reaching out to experts for Human Practices purposes. Oversaw transformation of GFP from kit plate.

**Katie:** Attended a SURF seminar about performing a literature review. Contacted the on-campus experts Dr. Sors recommended about the creation of a lung model, Dr. Gert Breur from the Purdue Veterinary School and Dr. Andrea Kasinski from the Purdue Department of Biological Sciences. Received replies from Dr. Breur and his colleague as well as from Dr. Kasinski and her post-doc and graduate student pertaining to their donation of rat lungs to the project. Conducted a literature search in order to evaluate the lung model methods. Looked into pricing for the decellularization/recellularization bioreactor creation.

**Kevin:** Began more extensive research on the promoter preceding the atpI gene, including looking at conserved regions of its predicted promoter sequence amongst different species of bacteria. Of these bacteria, there was a consensus region of approximately 30bp. Using the relative frequency of base pairs from a weblogo plot as well as a random number generator, 20 possible new universal promoters were designed. All promoter sequences were run through a promoter predictor algorithm and the 12 that were recognized and given a score progressed to the final test. From these 12 promoter sequences, 5 were chosen based on their scores and how conserved the -10 and -35 consensus regions were.

**Vi Ka:** Looked into logistics for culturing bacteria from lung swab; made a spreadsheet on common genera of bacteria from the lung microbiome, optimal growth temperature/media, type (gram-positive/gram-negative), and incubation time.

## Wednesday, 5/31 Day 7

**Andrew:** Python script was constructed based on parameters from given reference from Dr. Rickus. Enzyme kinetics and reactions rates in the context of modeling were reviewed for use. Research on using the Michaelis-Menten equation for modeling was done to propose as first method. Bacteria transformed from the previous day was inoculated as a review. Linear algebra applications to metabolic modeling were found, including flux balance analysis to optimize closed system describing the metabolism.

**Archana:** Continued contacting companies for supply materials. Emailed companies requesting funding. Organized budget. Searched for lowest prices for supply orders. Attended human practices meeting with Dr. Umberger to discuss FDA approval regarding our project. Finalized and submitted Experimental Campaign for review.

**Caleigh:** More research into United States FDA regulatory processes that would affect our therapy. as well as the approval process. Drafted the survey to gauge public acceptance of our therapy. Arranged and attended human practices meeting with Dr. Geanie Umberger in regards to the FDA approval process.

**Katie:** Conducted a literature search and continued investigating the lung model options for tomorrow’s meeting. Attended a meeting with Dr. Umberger about FDA regulation for our product.

**Kevin:** Researched previous protocols on culturing cells in 96-well plates and wrote the protocol for determining the ability of different quantities of benzene to inhibit the growth of E. coli.

**Vi Ka:** Completed logistics for culturing bacteria from swab. Reviewed transport assay (contacted Molecular Devices company regarding Fatty Acid Uptake Assay Kit, and Thermo Fischer for Bodipy Fatty Acid Analog protocol). Started reviewing statistics/literature for at-risk populations from benzene exposure.

## Thursday, 6/1 Day 8

**Andrew:** Training in miniprep protocol of GFP-expressing E.coli was performed. A working approach to metabolic modeling was determined based on data that would be collected from enzyme assays and literature. A complete script was constructed before meeting with advisors that produced a concentration vs time graph with varying levels of concentration. Flux balance analysis (FBA) was suggested as another method for identifying bottlenecks in the pathway.

**Archana:** Oversaw miniprep protocol of GFP expressing E. coli. Updated budget. Placed order for restriction enzymes. Contacted Thermo Fisher and MilliporeSigma. Drafted email for Michelle Chutka, our Cook Biotech contact. Drafted reminder email to Dr. Cooper.

**Caleigh:** Oversaw miniprep of GFP-expressing E. coli. Organized presentation for meeting with advisors. Refined protocols for creation of specific constructs from our ordered gBlocks.

**Katie:** Trained in miniprep protocol. Completed slides for meeting with PI about analysis of lung model methods. Met with graduate student advisor, Stephen Miloro, to obtain advice about project. Had meeting with Dr. Rickus and Dr. Solomon in which lung modeling method was selected for team: Decellularization/Recellularization. Improved upon my literature search based on Stephen’s comments in preparation for my writeup of my literature review due next week for SURF.

**Kevin:** Finished benzene growth inhibition protocol. Double checked literature for validation that universal promoter requires a sigma-70 sigma factor. Created slides for advisor meeting. Began research on ways to analyze enzyme nahK which produces carbon dioxide including possibly using an Octet Red analysis system.

**Vi Ka:** Received advice during weekly meeting regarding alterations to current protocol for the benzene transport assay. Started looking into further human practice related literature/information on benzene and its effect on the general populace.

## Friday, 6/2 Day 9

**Andrew:** All parts for reactions in the metabolic pathway were entered into the Python script. Changing and unchanging concentration options for the model were added. Research was conducted on how to incorporate flux balance analysis into the model. More graphing features were included in the figures produced by the model. PCR was performed on all the gBlocks received.

**Archana:** Contacted Dr. Cooper regarding GSMS. Reached out to Monsanto and Cook Biotech regarding funding and mentorship. Updated budget. Contacted VWR regarding pipet tips.

**Caleigh:** Organized personnel and protocols for cloning. Continued working on drafting survey.

**Katie:** Performed a peer review of Wiki contributions. Edited literature search document according to Stephen’s notes and completed SURF modules. Created graphic for wiki on lung model selection.

**Kevin:** Started the day by trying to calibrate pipettes that could potentially be used in later experiments and assays to find that our balances were not calibrated. Contacted Colleen Riley for tips on calibration. Contacted Dr. Rickus about use of the Octet-Red. Tried to find substrates that could be purchased so as to test our enzymes. Contacted Dr. Ma about using his lab’s Octet-Red machine and learned that it cannot be used to analyze catalytic enzymes. Made agar with LB broth and poured plates. Worked on universal promoter wiki page.

**Vi Ka:** Continued literature review on correlation between benzene and patients who fall ill/get sick. Created master mix of G-block at 1pg/ng, and DNA primers at 10mM; these were used for the following PCR protocol.

## Saturday, 6/3 Day 10

**Archana:** Oversaw Katie run PCR gel check on taq polymerase and Q5 PCR products. Wrote out instructions on how to use thermocycler.

**Katie:** Ran a quick gel check on the taq polymerase and Q5 PCR products. Determined that the Taq polymerase bands were not of the expected sizes.

**Vi Ka:** Determined that polymerase bands were wrong.

## Sunday, 6/4 Day 11

**Katie:** Re-did the PCR Preparation/Amplification protocol with Q5, as we ran out of Taq polymerase, changing the elongation time from 30 seconds to a minute and 30 seconds. Performed a quick gel check on the PCR products.

**Vi Ka:** Re-did the PCR Preparation/Amplification protocol with Q5, as we ran out of Taq polymerase, changing the elongation time from 30 seconds to a minute and 30 seconds. Performed a quick gel check on the PCR products.

[Back to Top](#top)

## Week 3

## Monday, 6/5 Day 12

**Andrew:** A .txt file reader was added to the metabolic model to read off data already given. The appropriate columns and rows in the E. coli core model were appended and modified for the new reactions present in the pathway. The framework for FBA was established using linear algebra methods and a brute force method of linear programming. The COBRA package for Python was downloaded to provide further possibilities.

**Archana:** Contacted MilliporeSigma and Thermo Fisher for quotes/discounts. Bought 96 well plates and Taq polymerase from Lilly store. Placed order for 1000 uL pipet tips from VWR. Completed payment details for Experiment campaign. Researched companies and emailed them for funding. Oversaw thermocycler use. Found QlAquick Gel Extraction Kit Protocol.

**Caleigh:** Screamed on rollercoasters.

**Katie:** Attended a SURF panel discussion about careers in academia. Analyzed gel results from previous day. Uploaded data and conclusions to drive. Created a 1x TAE Buffer and ran PCR for GBlock 4. Completed RCR-CITI training modules about conflicts of interest, data management, and plagiarism.

**Kevin:** Used an analytical balance to determine whether or not an 8-channel pipette was usable (it isn’t) as the calibration problems on the balance seemed to have improved slightly. Scheduled Coleen Riley to properly calibrate the balance on Tuesday. Contacted multiple offices to try and arrange a human practices meeting with BP with little success. Updated benzene quantification protocol.

**Vi Ka:** Continued to work on PCR for G-block 4, since the resulting DNA band was at the wrong size. Continued to modify protocol for the transport of benzene, recalculated amount of solutions needed in order to run multiple assays. Added few more contacts to HP master list.

## Tuesday, 6/6 Day 13

**Andrew:** The stoichiometric matrix in the FBA model was adjusted not to include the consumption of benzene without an inflow of benzene to correct apparent infeasibility. Brute force linear programming fell short of working with constraints given (especially minimums). Wet lab work was performed including gel creation, gel running, gel imaging, and gel extraction.

**Archana:** Bought 10uL gel tips from Lilly Store. Filled and autoclaved tips and tubes. Ran gel for Q5 products, imaged, analyzed, and excised correct bands. Ran gel for #4 Q5 product and raw #4 DNA, imaged and analyzed gel. Performed gel purification on Q5 excised DNA bands.

**Caleigh:** Ate Mickey Mouse shaped food.

**Katie:** Autoclaved tips and tubes. Ran a gel on the Q5 PCR products. Analyzed results and excised correct bands. Started literature review and wrote an elevator pitch on the project for SURF.

**Kevin:** Updated benzene quantification protocol so that low concentrations could be achieved by dissolving benzene in small quantities (40uL) of ethanol. Ran a gel for the G4 DNA which failed. Purified DNA via gel extraction for G1, G2, G3, G5, and G6 DNA.

**Vi Ka:** Prepared PCR for all G-block products, ran at 57.1 celsius with annealing time of 30 seconds, and elongation time at 1.5 minutes. Analyzed gel, and excised band under blue light. Continued revision of protocol.

## Wednesday, 6/7 Day 14

**Andrew:** Another researcher (Adrian Ortiz) was contacted to provide input on metabolic modeling. After checking for bugs in the brute force Python coding for FBA, no success was found on identifying a non constraint violating linear combination of null space vectors to define the flux. The module COBRApy was applied under the E. coli core model with success concerning FBA/FVA, supplying bounds of fluxes for 85% max efficiency of benzene uptake. Work on connected the flux balance/variability analysis to enzyme kinetics continued as well as on substrate channeling.

**Archana:** Contacted MilliporeSigma and Thermo Fisher contacts regarding product shipments. Organized and uploaded timecards onto Drive.

**Caleigh:** Checked GroupMe every 5 minutes. Panicked when it has been complete silence for 30 minutes - like having kids, silence is never good.

**Katie:** Continued literature review. Wrote a summary of the week’s events for the end-of-the-week update. Prepared slides for Thursday meeting. Attended Graduate School Information seminar for SURF.

**Kevin:** Looked into different solvents that could be used to dissolve benzene but realized a solvent is not necessary for our experiment as benzene and toluene are soluble at the levels we are working with. Began work on making universal promoter wiki page more visually pleasing by using piktochart. Performed Gibson assembly on G-blocks 1-3.

**Vi Ka:** Prepared PCR product for G-block 4 again, ran it at 62.1 celsius with annealing time of 30 seconds, and elongation time at 1.5 minutes. Went over benzene protocol with a graduate advisor, and amended protocol accordingly.

## Thursday, 6/8 Day 15

**Andrew:** Research was conducted on the effect of gene regulation on metabolic models, especially on models using flux balance analysis. Different tests were run in the COBRApy environment to optimize various objective functions. An organized presentation of this progress was presented at our weekly meeting. Milk powder was obtained from another lab for bacterial preservation.

**Archana:** Booked flight tickets for summer team to attend Jamboree. Looking into hotel bookings. Updated budget.

**Caleigh:** Skyped in to our weekly meeting because I miss everyone. Waiting in lines is a lonely business.

**Katie:** Finished literature review. Attended weekly PI meeting and took notes. Attended SURF information seminar on becoming a professor.

**Kevin:** Performed Gibson assembly on the G1-G3 fragments to combine them on a pSB1C3 plasmid. Performed transformation with the product of this Gibson assembly.

**Vi Ka:** Drafted first email to be sent out to BP and other HP contacts, waiting for feedback on draft. Clarified amount of bacteria needed to put in each well plates for the transport assay of fatty acid - was suggested that I contact another graduate student on using the haemocytometer.

## Friday, 6/9 Day 16

**Andrew:** Protocol was edited once more for bacteria preservation to prepare for first run next week. A few milliliters of protective medium was created along with about a milliliter of LB broth containing competent E. coli. A test run was started with a sucrose/milk solution and a nanopure water solution. Data showed bacteria were viable after PBS wash and after an hour in -80 °C freezer.

**Archana:** Submitted payroll forms to Pam. Worked on Experiment campaign Facebook/Twitter blurbs and press release. Researched hotel bookings for Jamboree.

**Caleigh:** Said goodbye to Disney World! I will be back!

**Katie:** Revised literature review based on graduate student feedback. Edited the Experiment Campaign document. Wrote wiki content. Worked on social media plans.

**Kevin:** The transformation from the day before was not very successful as only a single colony was formed on the experimental plate. Performed PCR on colony. Helped remove gigantic spider from basement. Worked on universal promoter piktochart.

**Vi Ka:** Made amendments to press release form. Updated Experiment campaign before launch. Starting wiki:project description:lung microbiome.

[Back to Top](#top)

## Week 4

## Monday, 6/12 Day 17

**Andrew:** Viability checks from over the weekend were verified, seeing viability on all conditions after two days preserved. PCR was performed on all gBlocks to increase product concentration for pGEM later. More sucrose/milk solution was created to prepare for first full run of preservation protocol this week. Two additional tubes of competent E. coli were made to run this protocol and to test dilution factors for counting colony forming units. The entire method for protocol was summarized to smooth out logistics.

**Archana:** Shared VWR’s pipette donation on social media. Collected information for SAO Travel Form for Jamboree. Contacted Sheraton Hotel regarding pricing. Updated/organized budget. Took and provided feedback on survey. Searched for propane canister prices.

**Caleigh:** Autoclaved full biohazard bag and arranged for REM pickup. Edited survey draft to help improve readability. Drafted responses to IRB form for review exemption for survey.

**Katie:** Attended elevator pitch workshop for SURF. Created serial dilution of NADH for BnzABCD assay. Shared pipette tip donation and Experiment campaign on social media platforms (Facebook, Twitter, and Instagram). Created new wiki infographic. Annotated HP survey draft.

**Kevin:** Made and ran gel testing to test the presence of the correct gibson construct which failed. Worked on universal promoter wiki and began work on modifying the universal promoter so that the ratios of the constitutive organisms is more desirable.

**Vi Ka:** Sent out first email to d.porter of BP Whiting plant. Match experiment pledge with $51 (one dollar extra!) Redid PCR for G4, with reverse touchdown thermocycler setting, and double MgCl2 concentration: 1) G4 with Q5, 2) G4 with Q5, 2x MgCl2, 3) G4 with Taq, 4) G4 with Taq, 2x MgCl2. G4 didn’t work!

## Tuesday, 6/13 Day 18

**Andrew:** Viability checks from weekend were recorded and showed viability in all conditions. Each condition was plated again for checking viability. A digest was run for GFP in preparation for testing universal RBS strengths, created early on in the summer. Two LB plates were made to begin testing dilutions for proper dilution factor of CFU counts on actual protocol run. This is expected to begin before Friday of this week and terminate before July. A gel was then prepared for running the GFP digestion followed by loading a remaining PCR sample along with the GFP. Early bacterial growth on dilution plates showed definite decrease in CFU density, providing optimism for finding appropriate dilution factor.

**Archana:** Updated budget. Edited thank you note to backers. Provided feedback on Experiment campaign slide for Bindley lobby. Overlooking Biomakers website posts. Researching and emailing companies about propane canister discounts. Worked on press release. Called up Sheraton and ThermoFisher. Provided feedback on Biomakers website posts.

**Caleigh:** PCR amplification of all gBlocks, trying several troubleshooting procedures for gBlock 4. Finished drafting IRB exemption form for survey. Provided feedback on press release draft. Completed iGEM Safety Forms 1 through 3. Planned PCR troubleshooting strategies.

**Katie:** Wrote a thank you note to backers. Created a slide about the Experiment campaign for Bindley Bioscience Center lobby. Edited the Experiment Campaign Press Release. Wrote blog posts for the Biomakers website.

**Kevin:** Continued and completed newly designed universal promoters. Worked on graphs demonstrating the makeup of the promoters to help illustrate the design process. Performed gel purification and performed early steps of pGEM on g-blocks 1-3.

**Vi Ka:** Edited Experiment Campaign press release final draft. Designed infographic for ‘Lung Microbiome’ page on wiki. Started researching on whether bacterial species native to the lung microbiome may become infectious when they are in different microbiomes.

## Wednesday, 6/14 Day 19

**Andrew:** Gel extraction was run on the GFP and gBlock 6 PCR product. Dilutions were prepared for Gibson of GFP/universal RBS's and gBlocks 1-3. Gibson assembly was performed on all five universal RBS's with GFP to test strength. To continue the preservation protocol, viability tests were checked from the previous day to show viability continuing for all conditions of preservation. Then, viability checks were run on all conditions of preservation.

**Archana:** Met with Amber, lab manager for BIND 134. Worked on embedding piktocharts onto the wiki. Provided feedback on several Biomakers website posts. Helped Andrew with Gibson Assembly for RBS’s. Learning how to use bootstrap on websites for wiki.

**Caleigh:** PCR on gBlocks 4,5, and 6 using DMSO as an additive. Autoclaved pipettman tips, microfuge tubes, and glass beads. Helped plan for pGEM transformations and Gibson assembly. Contacted Indiana State’s Department of Health Tobacco Use Prevention and Cessation Commission in regards to a potential Human Practices meetup. Contacted University of Michigan as potential collaborators. Prepared mock survey to assess how long it takes people to complete. Performed touchdown PCR on gBlocks 4, 5 and 6 using DMSO, glycerol, or GC enhancer as additives.

**Katie:** Prepared and poured a gel. Met with Amber, the manager of lab BIND 134, to get a safety and general lab rundown. Wrote more blog posts and updates for the Biomakers website. Wrote thank you notes to Experiment campaign backers. Began the humans of purdue iGEM social media campaign

**Kevin:** Performed pGEM transformation, rewrote E. coli transformation protocol for clarity, worked on Universal Promoter wiki.

**Vi Ka:** Met with Amber, BIND 134 lab manager. Finished Piktochart for Project Description: Lung Microbiome. Ran gel for PCR products G-block 4, 5, 6, which were made using DMSO and imaged them - nothing worked. Sent emails to Josh Clark (Vet Tech Instructor), and Russell Lagsdon (LILY Media Prep), asking for donations of blood agar plate. Made the word ‘groovy’ great again.

## Thursday, 6/15 Day 20

**Andrew:** Viability tests were checked from previous day to find positive results from all conditions of preservation. Transformations on E. coli for RBS's with GFP were completed. Viability tests were run on all conditions of preservation to continue test run. The weekly meeting with advisers provided feedback concerning focusing on negative control of preservation protocol. Then the negative control on sucrose/milk mix was plated. A subsequent negative control on LB broth was run along with a positive control on cultured cells. This was followed by preparation of 44 tubes for the preservation protocol. Final edits were added to the protocol.

**Archana:** Updated weekly presentation slides, searched for good wikis that will resemble our 2017 format. Responded to Experiment backer, Dr. Singh. Provided feedback on Biomaker website posts. Worked on wiki. Sent emails to company contacts. Talk to Adrian regarding pGEM. Tried using old thermocycler.

**Caleigh:** Updated daily plans and weekly presentation slides. Attended meeting with Hakeem Abdul Wahab from statistics department to receive feedback on survey design. Updated GANTT chart and checked to ensure that project is on task towards meeting goals. Met with advisors in regards to project updates.

**Katie:** Updated the Biomakers website. Wrote thank yous to letters to backers. Wrote the Week 4 Newsletter. Attended a statistics consultation meeting and took notes. Met with Dr. Rickus about SURF assignment. Attended PI meeting, but left early to go to a SURF research seminar about converting plastic waste into pure polymers, fuels, and clean energy.

**Kevin:** Continued work on universal promoter. Growth found on G1-G3 pGEM transformation plates. Incoculated.

**Vi Ka:** Updated presentation slides. Ran PCR for new G-block 4 conditions. After today, we don’t need to do anymore PCR! Finished Lung microbiome piktochart.

## Friday, 6/16 Day 21

**Andrew:** The first iteration of the preservation protocol was started after checking all negative and positive controls. Dilution tests were plated after an hour of initial freeze for each condition. A volume of 500 mL of agar was created for 20 new LB plates. Viability checks were run on lines available for preservation conditions. Thank you emails were sent out to donors whom I know. An additional 22 LB agar plates were poured from the prepared volume.

**Archana:** Spoke to Susan regarding free Bindley supplies! Sent Pam packing slips. Responded to Larry, our Monsanto rep. about $ and meeting up and ThermoFisher regarding instrument trade-ins. Edited/reviewed Biomakers website posts and newsletter. Submitted SOGA Activity Form for Jamboree for approval. Searched up HiFi/Gibson Master Mix protocol and worked on ordering materials. Checked Solomon lab for materials. Searched online for supply pricings. Sent email to Piktochart regarding free premium account.

**Caleigh:** Contacted iGEM HQ, potential collaborations, and human practices connections. Finished updated GANTT chart to represent current project status. Redid calculations for Gibson assembly for RBS-GFP constructs.

**Katie:** Updated Experiment Campaign slide for Bindley and Biomakers blog. Finished the weekly email update. Posted to social media!! Contacted other Biomakers about Biomakers of Purdue Social Media Campaign. Completed the Social Behavior Research Training on CITI.

**Kevin:** Performed miniprep, collected concentrations, ran a gel, and excised bands for G1-G3. Only G3 showed results that weren’t promising. Re-inoculated G3. Began research on universal promoter via random mutagenesis.

**Vi Ka:** Performed NEB A-tailing with Taq polymerase. Used the wrong buffer the first time, had to redo the procedure. Completed procedure in the afternoon and proceeded with pGEM ligation. Left ligation products to sit overnight in 4 celsius.

## Saturday, 6/17 Day 22

**Andrew:** Dilution factors were verified from cultures thawed after an hour. The new LB plates were labeled that had been poured the previous day. Day 1 solutions from the preservation protocol were plated twice due to worries about the amount of diluted solution on the plate spreading. Test run tubes from the preservation protocol were plated as long as solution volume remained. A results spreadsheet was added to the results folder under bacteria preservation.

**Caleigh:** Miniprep of gBlock 3 in the pGEM T-easy vector. Digestion of a GFP from kit plate and gel purification of the gene. Bacterial transformation of the gBlocks 4-6 pGEM ligations.

## Sunday, 6/18 Day 23

**Andrew:** Preservation protocol tubes were plated along with preservation protocol test run tubes.

**Caleigh:** Took pictures of bacterial plates for Father’s Day social media post.

[Back to Top](#top)

## Week 5

## Monday, 6/19 Day 24

**Andrew:** CFU counts for ¾ plates were recorded from previous day. Dilutions from failed plates were redone to get CFU data. Preservation protocol tubes were then plated for the present day. Two new graphs were added into the Google sheet to track data over time. Ligation on the lambda digest followed to help check if ligation was working. PCR purification on gBlocks 4-6 was performed with issues of pH level. Test run preservation protocol tubes were plated.

**Archana:** Looked and sorted through free Bindley supplies. Looked through lab drawers for extra materials. Reviewed and edited PurdueBiomakers.com post. Calculated amount of Gibson reagents required. Emailed Dr. Solomon for Gibson Assembly master mix clarification. Called companies for discounts. Asked Solomon lab for materials. Sent Katie Social Media Campaign information.

**Caleigh:** Picked up free supplies and organized the lab in order to accommodate them. Planned troubleshooting procedures for failed pGEM ligations and transformations and discussed ideas with the graduate student Kok and Dr. Solomon. Contacted REM about disposal of old used mineral oil. Planned for team to attend a discussion about the current state of cancer research as a Human Practices event. Planned and delegated labwork for the day, answering questions and helping as needed.

**Katie:** Attended SURF Abstract Workshop. Scrounged for bioreactor parts in the free Bindley giveaway table. Updated Biomakers Blog. Made X-Gal Plates. Worked on social media campaign.

**Kevin:** Enjoyed actually seeing sunlight all day.

**Vi Ka:** Started work on project description piktochart. Prepared digestion for Lambda DNA, PSB 1C3, and G3 block. Accidentally left products in gel for too long - image was not clear. I WILL STEP UP MY GAME.

## Tuesday, 6/20 Day 25

**Andrew:** CFU counts were recorded for preservation protocol plates from the previous day. Preservation tubes for present day were then plated. iGEM Toulouse was contacted and was open to collaboration. Katie received info for the researcher feature. A search for units used for FBA/FVA was started to apply to the mathematical model. Gel extraction was performed with poor results concerning DNA concentration. GFP transformed bacteria were inoculated for digesting the next day. Viability tests were checked from the previous day with positive results. Test run preservation protocol tubes were plated.

**Archana:** Calculated reagents needed for Gibson Assembly reagents and did cost analysis for products. Talked to Dr. Solomon and Kok regarding products and prices. Emailed Katie Maish and Dr. Rickus regarding payroll. Had conference HP conference call with TUPCC. Researched ethanol prices. Completed CITI Training. Drafted email to Dr. Fernandez regarding a summer update meeting.

**Caleigh:** Arranged for REM to pick up used mineral oil. Planned and delegated lab work for the day. Finished CITI training for human subjects research for Human Practices Survey. Researched firefighter’s exposure to benzene and contacted the local fire department for a Human Practices meeting. Had a conference call with the Indiana State Department of Health’s Tobacco Use Prevention and Cessation Commission.

**Katie:** Messaged Team Pakistan on Facebook about a Skype call. Performed RFP and pGEM transformations. Took notes at HP conference call with Tupac. Attended SURF Research Seminar about Cyber Forensics

**Kevin:** Slept for 13 hours. Cried because all the jelly beans & cookies were eaten.

**Vi Ka:** Failed to step up my game. Performed miniprep on G-3 bacteria inoculation culture. DNA yield was only 2.1ng/uL. Fear not, tomorrow will be the day! Continued work on piktochart.

## Wednesday, 6/21 Day 26

**Andrew:** CFU counts were recorded for preservation protocol plates from the previous day. Preservation protocol tubes were then plated for the present day. Viability tests were checked from the previous day with one negative result on line 6. Test run preservation protocol tubes were plated after. Collaboration with iGEM Toulouse continued with intentions to Skype. A gel was run on gBlocks 1-3 and GFP gene from digestion. The method for numerical integration inside the mathematical model of enzyme kinetics was rewritten to be sensitive to large changes within a small time step.

**Archana:** Tried to apply for discounted education pricing from Piktochart. Emailed Pam for 501(c)(3). Tracked down Thompson Lab in search of T5 Exonuclease. Checked up on SOGA Jamboree Event status. Proofread Katie’s email to Dr. Ott. Edited Press Release. Worked on wiki/piktochart formatting.

**Caleigh:** Planned and delegated daily labwork. Worked on developing protocol for testing nebulizer and began preliminary testing. Worked on presentation for weekly updates meeting. Contacted IU Health and Franciscan Health for sake of Human Practices. Contacted Imagination Station (local children’s science museum) about potential public engagement event. Began research as to the need to degrade benzene and toluene for space travel.

**Katie:** Contacted euthanization lab about rat lung specifics. Drafted an email to Dr. Ott about tips for decellularization. Made LB Agar plates. Made slides for Thursday meeting. Sent Dr. Ott’s email. Photographed and inoculated the transformed colonies. Started on Human Practices Homepage wikis.

**Kevin:** Enjoying a leisurely time at home, drinking red bull. I miss the sweet, sweet smell of E. coli in the morning - hungry now.

**Vi Ka:** Performed miniprep on G-3 and GFP, with Epoch kit. Achieved yield of 62.7 ng/uL for GF-3, and 144.7 ng/uL for GFP. Prepared digestion for G-3, GFP, G-1, G-2. Worked on presentation for weekly updates meeting. Sent follow-up email to Donald Porter (BP); and new emails to John Companik (BP) and Tom Keilman (BP).

## Thursday, 6/22 Day 27

**Andrew:** CFU counts were recorded for preservation protocol plates from the previous day. Preservation protocol tubes were then plated for the present day. Viability tests were checked from the previous day with the one negative result on line 6 becoming positive. Test run preservation protocol tubes were plated after. The experiment was updated into a presentable for the weekly meeting - this included the suggestion of adding error bars to figures made from the data. A gel was then run on gBlocks 2-6 to purify more DNA. The gel was imaged and proper bands were extracted.

**Archana:** Submitted sample request for Zymo gel extraction kit. Edited Week 5 Newsletter. Worked on wiki formatting. Contacted Sam for NAD.

**Caleigh:** Worked on wiki content. Researched potential of our project being beneficial in space-exploration. Updated presentation slides and hardware testing protocol.

**Katie:** Performed gel extractions that did not turn out well at all :(. Wrote Week 5 Newsletter. Attended weekly PI meeting and got even more recommended changes for the lung model methods.

**Kevin:** Got tan lines that weren’t from the dim fluorescent lights in lab.

**Vi Ka:** Contacted Helsinki team regarding wiki coding. Performed miniprep on G3(1), G3(2), G3(3), G4, G5, G6, RFP. Obtained >100ng/uL for all except G3(3).

## Friday, 6/23 Day 28

**Andrew:** CFU counts were recorded for preservation protocol plates from the previous day. Preservation protocol tubes were then plated for the present day. Viability tests were checked from the previous day with one negative result on line 6. Collaboration continued with iGEM Toulouse. A gel was run on gBlocks 1-3 and GFP gene from digestion. The method for numerical integration was changed inside mathematical model of enzyme kinetics to be sensitive to a large change within a given time step.

**Archana:** Turned in payroll forms to Pam. Asked Pam about Vi Ka’s deducted pay and emailed Pam about it. Contacted Thompson Lab for T5 Exonuclease, they do not carry it. Called fuel cartridge vendor for discounts with no luck. Sent Pam order request for fuel cartridges. Worked with Solomon Lab to get feedback on our weekly presentations. Worked on wiki page edits.

**Caleigh:** Worked on wiki content for pathway design. Planned and delegated daily labwork. Picked up NAD+. Ran first run-through of protocol to test nebulizer. Performed digestion on pGEM plasmids to extract gBlocks.

**Katie:** Performed minipreps on gBlock 1, iterations 1,2,3. Escaped the dungeon early.

**Kevin:** Enjoyed my last day of paid vacation.

**Vi Ka:** Performed gel extraction - did not label spin columns, will have to come in tomorrow to re-run gels and identify the unknowns. Identified issue with time card - will give the time card to Dr.Rickus tomorrow to sign.

## Saturday, 6/24 Day 29

**Andrew:** CFU counts were recorded for preservation protocol plates from the previous day. Preservation protocol tubes were then plated for the present day.

**Vi Ka:** Ran gel for Unknown 1-6, and G-block 1-3, 1-2, 1-1. Excised bands for all G-blocks.

[Back to Top](#top)

## Week 6

## Monday, 6/26 Day 30

**Andrew:** CFU counts were recorded for preservation protocol plates from the previous day. Preservation protocol tubes were then plated for the present day. A video call with iGEM Toulouse was organized and held for an hour before lunch about collaboration ideas. A gel was made and run for GFP/gBlocks 2-3. CTI training for human practices work was completed.

**Archana:** Researched material pricing. Had Skype call with Toulouse!! Created piktochart for Team History. Emailed Piktochart for discount on account subscription. Payed and upgraded Piktochart account. Updated budget. Finished editing Press Release, contacted Paige to see how to send it out. Uploaded gif onto wiki.

**Caleigh:** Planned and delegated daily labwork. Continued work on creating wiki content. Made LB-amp plates and autoclaved more eppendorf tubes.

**Katie:** Attended a SURF seminar about conveying research to a lay audience. Wrote the Biomakers Blog weekly update. Skyped with Toulouse & posted about it on social media. Edited Experiment Press Release. Emailed How It Works about feature.

**Kevin:** Completed CITI Training, labeled conical tubes and inoculated E. coli in preparation from a trial run of the MIC50 assay, emailed Bruce Cooper about training for the GC, wrote biography for the wiki and humans of Purdue, researched potential error-prone PCR methods to use in generating a universal promoter, and ran a G2,G3, and GFP gel and extracted.

**Vi Ka:** Performed miniprep for G-block 2, GFP. Obtained results for G-block 2 with 529.9 ng/uL DNA, and GFP with 126.4 ng/nL. Prepared digestion mix for G-block 2, G-block 3, and GFP. Finished Piktochart for Project Description (now aptly renamed to: Ez Breathe-y). Edited Piktocharts of Lung Microbiome and Ez Breathe-y per feedback.

## Tuesday, 6/27 Day 31

**Andrew:** CFU counts were recorded for preservation protocol plates from the previous day. Preservation protocol tubes were then plated for the present day. Pictures were taken for newsletter and headshots. Extensive work was started on Wiki pages in Piktochart for topics assigned like bacteria preservation. Gibson assembly was performed on RBS 1 with GFP and also the lower operon of gBlocks 4-6.

**Archana:** Delegated Mark’s DoG hand-written note to Katie. Worked on Press Release final! edits. Sent companies emails for discounted Gibson products and ethanol. Worked on wiki. Went to Lilly Store to return 96 Well Plates and ABE to sort out salary issues.

**Caleigh:** Planned and delegated daily labwork. Worked with Andrew on bacterial preservation protocol. Provided feedback on teammates’ wiki content. Worked on wiki content. Met with West Lafayette Fire Department for a Human Practices meeting.

**Katie:** Contacted Valencia about Skype/collaboration. Finished catching up on Social Media thing. Edited press release. Went to Fire Department HP Meeting. Made PI meeting slides. Contacted Dr. Ott through his contact us page. Worked on Silver and Gold HP wikis.

**Kevin:** Gel purification for all G-blocks except G3, research on universal promoter. Recorded day 0 OD600 of benzene growth inhibition assay.

**Vi Ka:** Performed miniprep for G3-1 (158.2ng/uL), G3-2 (159.3ng/uL), G3-3 (90.4ng/uL). Made two digestions per G-block. Prepared gel for digestion PCR products. Worked on Piktochart for advice. Talked with SCU-China regarding collaboration/work on wiki content. Received advice from Aalto-Helsinki’s for wiki editing as well.

## Wednesday, 6/28 Day 32

**Andrew:** Enjoyed actual sunlight in Millennium Park. Couldn’t get over not counting CFUs, so counted blades of grass in the park, which held the dearest Azospirillum brasilense.

**Archana:** Compared supply quotes from company responses. Updated budget. Edited Weekly Meeting slides. Uploaded, edited, formatted Running Lab Notebook onto wiki. Uploaded Project Description Piktochart onto Wiki. Contacted Paige regarding Press Release.

**Caleigh:** Planned and delegated daily labcwork. Diluted and plated preserved bacteria. Contacted various other iGEM teams. A-tailed gBlock 3. Worked on wiki content. Inoculated gBlocks 1,2, 4, 5, and 6.

**Katie:** Finish draft of technical paper. Worked on slides for tomorrow. Performed E. Coli transformations using RBS 1 GFP and lower operon Gibson and plated. Edited piktocharts based on peer review.

**Kevin:** Performed TodX ligations on G1 and G4 (unsuccessful). Recorded day 1 OD600 of benzene growth inhibition assay. Worked on benzene wiki page.

**Vi Ka:** Spoke with Jonathan regarding updating the homepage design title to: Ez Breathe-y, with subtitle: Engineering the Lung Microbiome to Degrade Benzene and Toluene. Requested a design icon/picture of a human breathing in benzene into the lungs. Will check in with him 2 weeks from now. Continued work on Piktochart. Prepared PCR mix from G-block 3. Performed gel extraction and excision of bands - only G3-1, G3-2, G3-3 were visible; G1 and G4 were not.

## Thursday, 6/29 Day 33

**Andrew:** CFU counts were recorded for preservation protocol plates from the previous day. Preservation protocol tubes were then plated for the present day. NCI talk was attended along with the weekly lab meeting.

**Archana:** Worked on Press Release, compared prices for supplies. Attended presentation by Dr. Douglas Lowy, Acting Director of the National Cancer Institute, regarding cancer research. Emailed Dr. Rickus regarding gas canister order approval. Edited weekly presentation slides.

**Caleigh:** Worked on presentation for weekly meeting and wiki slides. Planned and delegated labwork. Attended a presentation by Dr. Douglas Lowy of the National Cancer Institute.

**Katie:** Edited the Experiment Press Release. Attended the SURF Technical Paper Paper Peer Review Session. Attended the National Cancer Institute seminar and got to meet Dr. Douglas Lowy, the organization’s director!! Attended the weekly PI meeting.

**Kevin:** Recorded day 2 OD600 of benzene growth inhibition assay and updated protocol to improve future results.Gel purification of G-blocks 1-3.

**Vi Ka:** Mentally and physically prepared to perform 10, TEN minipreps today with Kevin. Had an enlightening 20 minutes all alone (re:stuck) in the lift as I contemplated the meaning of life (a.k.a the existence of wifi). Completed transformation for G-block 3 (ligation done by Katie on June 28th). Made amendments to Piktochart (Lung Microbiome). Sent email asking Dr. Rickus and Dr. Solomon for help regarding obtaining blood agar plates from Tim Strong. Asked Meo from Aalto-Helsinki for advice regarding HTML/CSS coding.

## Friday, 6/30 Day 34

**Andrew:** Recorded CFU counts for plates from two days ago. Plated preservation protocol tubes for present day. Transformed E. coli with upper operon and plated. Reviewed and cleaned up mathematical model to create framework for full-scale model. Looked to include enzyme production/degradation in mathematical model. Sent out weekly update email.

**Archana:** Called up and emailed companies for supplies. Researched supply prices. Worked on wiki formatting. Drafted email for press release. Participated in Skype call with iGEM Team in Washington University in St. Louis. Sent out Press Release!! Sent Kathy/Pam and email regarding transfer of funds. Participated in Skype call with iGEM Team in Michigan State University. Edited week 6 newsletter. Emailed Piktochart for reimbursement receipt. Deleted template information off of wiki pages.

**Caleigh:** Assigned and delegated daily labwork. Participated in Skype calls with Washington University in St. Louis and Michigan State University’s iGEM teams. Uploaded content to wiki. Drafted introduction and questions for our email exchange with Pioneer Oil.

**Katie:** Posted to social media about the NCI talk. Edited Wiki Piktocharts for HP. Wrote Week 6 Newsletter. Died because the NATIONAL CANCER INSTITUTE LIKED, FOLLOWED, AND REPOSTED THE PURDUE BIOMAKERS INSTAGRAM POST THAT SHE MADE!!

**Kevin:** Digested, ran gels, and extracted G1, G2, G4, G5, and G6. Performed Gibson assembly for the upper operon (G1-G3) and transformed. Found potential RBS for Shewanella oneidensis.

**Vi Ka:** Checked on G-block 3 transformation. Nothing worked (P.C, B.C, G-3). Made 5 PCR mix for G-block 3. Edited Wiki Piktocharts. Started wiki for Unmet Needs and Target Population (tentatively)

## Saturday, 7/1 Day 35

**Caleigh:** Calculated titer of Andrew’s preservation protocol. Performed colony PCR on two colonies from Gibson transformation. Gel electrophoresis of colony PCR products, gBlock 3 PCR products, and gBlocks 1, 2, 4, 5, and 6 pGEM digestions.

## Sunday, 7/2 Day 36

**Caleigh:** Performed Gibson assembly on one universal RBS and upper operon. Digested Gibson products. Performed PCR purification to increase concentration of gBlock 4.

[Back to Top](#top)

## Week 7

## Monday, 7/3 Day 37

**Andrew:** Sat on a beach, wishing to be in a grassier place with my beloved Azospirillum brasilense. Sand bacteria just isn’t the same.

**Archana:** Chicago’s sunlight > basement. Also lowkey salty that everyone finished the chocolate chip cookies and brownies without me.

**Caleigh:** Assigned and delegated daily labwork. Autoclaved supplies and biohazard bags. Emailed in order to get Mycobacterium smegmatis. Contacted leukemia and lymphoma society.

**Chris:** Joined the iGEM team. Completed literature research on benzene respiration data for different environments, adsorption data of inhalation, and OSHA benzene regulation requirements.

**Katie:** Am glad to be home because people can finally enjoy my puns. D-1: Already missing my Twitter game. Spent 40% of my day laughing because I still don’t follow Kevin back on twitter.

**Kevin:** Digested G1,G2, G4-G6 pGEM inserts (failed!!!). Miniprepped G1, G2, G4-G6. Digested again.

**Vi Ka:** Did transformation for Operon ligation, RBS digest (with ligation), and RBS with no ligation. Excised PCR products, g-3; others did not work out. Helped Kevin out with parts of the miniprep - I heart miniprep!

## Tuesday, 7/4 Day 38

**Caleigh:** Performed colony PCR with a heart full of hope. Created wiki content. Poured LB and LB-chlor plates. Gel electrophoresis of colony PCR and digestion products. Hope was crushed.

**Kevin:** Ran a bunch of gels that ended up not working. Uploaded universal promoter designs to Benchling and decided to include the original promoter and the original promoter with more spacing between the -10/-35 regions as additional promoters to test. Tested viability of Invitrogen cells.

**Vi Ka:** Prepared miniprep for G1 - G2, G4 - G6, and GFP. Prepared digestion mixture. Achieved excellent results, 116 < results < 620.

## Wednesday, 7/5 Day 39

**Archana:** Updated running budget. Fixed some email issues. Resent emails to companies for quotes, Pam/Kathy for donation transfers. Tried resending press release emails, but they bounced. Updated Biomakers Twitter banner. Emailed Dr. Fernandez to set up meeting. Chatted with A Small Orange customer service regarding purduebiomakers.com payment. Edited reimbursements and budget pages and emailed Dr. Rickus. Worked on wiki.

**Caleigh:** Discussed troubleshooting for cloning attempts with our graduate advisor, Kok. Worked with Archana and Katie to develop a plan to revitalize Experiment campaign. Created wiki content.

**Chris:** Downloaded and viewed tutorial on Bootstrap template code to implement in wiki. Established local server to test Bootstrap and html pages.

**Katie:** Poured a 1% agarose gel. Skyped with SVCE Chennai. Hyperventilated about call with Dr. Ott. Called Dr. Ott and found out that we can’t decellularize a lung for our purposes, but got a lot of useful ideas and feedback!!! Emailed Dr. Rickus about this design change to get her thoughts. Emailed How It Works again. Edited slides for tomorrow’s meeting. Worked on Piktocharts for wiki. Updated the Biomakers blog.

**Kevin:** Called Dr. Ott with Katie. Discussed how to proceed with cloning and identified errors in miniprep procedure with Kok and Dr. Solomon. Ran PCR for G-blocks 1-3, 5-6.

**Vi Ka:** Identified potential errors in miniprep protocol with graduate advisor, Kok. Worked on wiki homepage.

## Thursday, 7/6 Day 40

**Archana:** Worked with Katie to write interview question responses to How It Works magazine. Edited weekly meeting slides. Worked on writing Experiment.com lab note. Called and set up appointment with Dr. Harris for tomorrow, and emailed his secretary. Sent Pam order for Taq and T5 exonuclease. Updated Supply Order List.

**Caleigh:** Planned and delegated daily labwork. Worked with Archana and Katie to brainstorm ideas as to what to say for How it Works responses. Emailed about gaining access to certain equipment. Finished miniprep after Vi Ka had to leave.

**Chris:** Discussed with Andrew regarding modeling and how to implement. Read article regarding Bootstrap styling and learned how to implement horizontal menu dropdown bar

**Katie:** Performed a DPNI digest. Emailed How It Works. Edited slides. Taught Chris how to do gel purification and Nanodrop’d the products. Went to lab meeting.

**Kevin:** Gel purification, learned how to make competent cells, attended lab meeting, inoculated to make competent cells for tomorrow

**Vi Ka:** Ran PCR products for g-block 1,2,3 (x3), 5, 6; excised bands for all products except g-block 1. Did first part of miniprep for g-block 4, up to adding P2 lysis buffer. Made digestion mixture.

## Friday, 7/7 Day 41

**Andrew:** The living E. coli was surprised out of the team because I came back early. All old preservation protocol tubes were removed from freezers. The modeling team was contacted about future directions and meeting next week. Modeling action items were created for the modeling meeting. Chris was helped with the nanodrop. The preservation protocol was rewritten.

**Archana:** Turned in time cards to ABE, discussed shipping concerns, and requested account statement. Emailed Pam about incorrect shipment. Sent out all press release emails. Submitted our press release to BioSpeakIndiana. Edited weekly newsletter. Attended meeting with Dr. Harris to secure funding from College of Engineering.

**Caleigh:** BLAMES VI KA FOR STUFF SHE DID NOT DO (aka writing weird stuff on white board). SAD. VI KA PLEADS INNOCENCE FOR NOT WRITING NERF WARS ON THE BOARD. Skyped with University of Iowa. Attended a meeting in regards to funding.

**Chris:** Gel Purification for gBlock 4. Conducted nanodrop test for gBlock 4

**Katie:** Gibson’d the upper operon. Finished technical paper. Write the weekly newsletter. Skyped with the University of Iowa (not Iowa State) and posted on social media. Messaged with team Peshawar.

**Vi Ka:** Prepared digestion for g-block 4 (x 4) from last yesterday’s miniprep. Ran PCR for the digestions and excised the bands.

[Back to Top](#top)

## Week 8

## Monday, 7/10 Day 42

**Andrew:** PCR was performed on gBlocks 1-3. All 42 preservation tubes and 2 dilution check tubes for the next preservation protocol were labeled. Protective medium was then prepared for the protocol and negative controls were plated. Wild-type E. coli were inoculated to grow overnight for the protocol. A digest was run of gBlock 4 for TodX transoporter. iGEM Toulouse was contacted as a follow up. Purification of gBlock 4 digest for TodX was completed.

**Caleigh:** Planned and delegated daily labwork. Contacted potential HP connections. Skyped with Peshawar and Valencia. Organized collaborations.

**Chris:** Scheduled meeting with Andrew and Vyaas for mathematical modeling. Created LB-agar solution for plating. Read up on InterLab Study 2017 documentation in preparation of plate reading.

**Katie:** Skyped with Team Peshawar. Attended SURF seminar about giving an oral presentation. Skyped with Valencia! (A Katie le encanta hablar en español). Posted to social media about the Skypes. Wrote the weekly Biomakers Blog. Emailed Dr. Rickus about new experimental design. Left early because she had to adult and run errands.

**Kevin:** Gel of colony PCR and Gibson, PCR check of Gibson, inoculated LB in preparation for MIC50 assay, and tested electrocompetent cell transformation efficiency

## Tuesday, 7/11 Day 43

**Andrew:** The negative control for preservation protocol was checked, finding a positive result on protective medium. The prepared protective medium was boiled for a minute over open flame by bobbing up and down over it. A random sequence of numbers was obtained for plating the protocol. Different methods for boiling the protective medium were tested. Work on expanding parts of the mathematical modeling was conducted, and action items were organized for the meeting.

**Archana:** Emailed Lori Leroy of BioSpeakIndiana regarding Press Release. Sent emails regarding donation transfers and supply orders. Emailed Susan about air vent leak. Provided feedback on Experiment campaign note about firefighters. Searched for supplies. Worked on Wiki code.

**Caleigh:** Planned and delegated daily labwork. Skyped Rose Hulman Institute of Technology. Wrote a note for Experiment campaign. Created wiki content.

**Katie:** Ate Vi Ka’s brownies. They were delicious. Emailed Dr. Rickus about experimental design again because Purdue email sucks. Worked on presentation slides draft. Ate more brownies. Edited Experiment note. Shopped for DEALZ on Amazon Prime Day at lunch. Talked with Paul about lung model preparations. Hyperventilated about the lungs coming in one week. Emailed a bunch of people about the lungs. Skyped with Rose-Hulman. Attended Purdue Foundry seminar for SURF.

**Kevin:** PCR of gBlocks, ran some gels probably

**Vi Ka:** made thin brownies - brownies should be fluffy like bunnies! Glad katie likes the brownies. Ran gel for g-blocks + excise bands.

## Wednesday, 7/12 Day 44

**Andrew:** Negative control was checked for preservation protocol, finding positive on LB broth. Work continued on mathematical model with incorporation of FBA/FVA connection to glucose uptake and of cell growth in tandem with benzene degradation.

**Archana:** Updated Supply Order List. Worked on wiki code. Searched for supply costs. Called United Steelworkers and spoke to them about their opinions and concerns about our project.

**Caleigh:** Planned and delegated daily labwork. Emailed Human Practices connections. Placed order for universal promoters with IDT. Created wiki content

**Katie:** Edited ppt slides for SURF. Attended meeting with Stephen for feedback on the slides. Prepared for use of flex station. Used flex station under supervision of Suraj. Made a 1M solution of Tris-HCl. Called the largest manufacturing union in the country.

**Kevin:** Set up benzene growth inhibition assay, made gibson master-mix, and performed gibson assembly on the upper and lower operons

**Vi Ka:** Performed digestions for BnzABCD, TodX, and subsequently ran the gels. Also prepared PCR for G-blocks, and miniprep for G4. Obtained results of 1200ng/uL.

## Thursday, 7/13 Day 45

**Andrew:** Mathematical modeling was continued with completion of cell growth coupled with benzene degradation for cell groups. Graphical representations of the data were implemented.

**Archana:** Dropped off forms to ABE, picked up pipette tips from Lilly Store. Emailed Monsanto about how to make donation. Asked Lori LeRoy from BioSpeakIndiana to retweet our tweet. Reviewed/edited weekly presentation slides. Researched other sites to run our blog on. Researched FBS and Nutrient

**Caleigh:** Performed a bacterial transformation at 2 am. Prepared presentation for weekly lab meeting. Troubleshooted cloning with graduate student advisor Kok. Wrote the first round of our title and abstract.

**Katie:** Set up, ran, imaged, and excised a gel of gBlock 3 PCR products. Prepared PI weekly meeting slides. Prepared SURF slide draft. Attended weekly meeting. Made plan for lungs. Delegated work to others to help with lung plan.

**Kevin:** Did a Gibson PCR check for previously Gibsoned upper and lower operons, cleaned up the lab, performed TodX G4 Digest, continued growth inhibition assay.

**Vi Ka:** Ran gel for Gibson PCR, started transport assay for growth and induction to collect cells.

## Friday, 7/14 Day 46

**Archana:** Went to pick up ventilator with Katie. Visited Imagination Station and discussed activity ideas and budget, and took meeting notes. Emailed companies to order gloves. Requested FBS sample from VWR. Searched for Ham’s F-12 Nutrient Mix prices. Called VWR to organize samples

**Caleigh:** Did gel extraction. Went to Imagination Station to discuss public engagement activity. Discussed possibility of using a vibratome. Ran a gel from colony PCR products.

**Katie:** Emailed Cherry about the ventilator. Emailed Ethan about sequencing and gas clamps and regulator. Got the ventilator. Emailed Dr. Rickus about microscopes and CO2 tanks. Wrote the weekly newsletter. Emailed. Solomon about hood and gas details. Got Starbucks free tea. Emailed a bunch of people and got some Ham F-12. Tried to get FBS sample from VWR.

**Kevin:** Performed BspEI digestion on SacII digested G4, continued benzene growth inhibition protocol, visited Imagination Station and set up an outreach event for next Saturday, performed colony PCR for the upper and lower operons, PCR to check that the Gibson for these operons worked, and PCR amplification for G1 and G2

**Vi Ka:** Continued transport assay for cells - O.G batch had a very high O.D of 1.020, Kok’s batch had relatively low O.D of 0.060. Made digestion mixture for G-block 3. Finished new draft for Homepage. Made a Lung Microbiome page.

## Sunday, 7/16 Day 47

**Caleigh:** Ran a gel from the PCR of Gibson products. Gel extracted and performed a Gibson reaction for BnzABCD and TodX constructs.

[Back to Top](#top)

## Week 9

## Monday, 7/17 Day 48

**Andrew:** Began second iteration of preservation protocol with negative controls remaining negative and cells grown overnight. Fixed accuracy of model such that calculations in model accurate within 1/100 of a percent. Started implementing competition into mathematical model. Created separate growth functions to change type of model.

**Archana:** Picked up labeling tape from Lilly Store. Emailed VWR with information to get FBS shipped tomorrow. Purchased new domain at Jimbdo. Copied over all information from website to transfer to new domain. Edited Experiment note to last year’s backers. Updated budget. Added comments to Alumni Donation Request document.

**Caleigh:** Tried to gain access to a vibrotome or microtome for organotypic culture. Planned and delegated daily labwork. Arranged for Pipettman calibration. Wrote an Experiment labnote to encourage backers of last year’s project to back this year’s project.

**Chris:** Transformed the suspended DNA for the interlab study.

**Katie:** Emailed a bunch of people about using a vibratome for the lungs. Performed a DNA extraction, PCR, and gel with Ethan as a dry run/control for the lungs.

**Kevin:** Made more LB chlor plates, transformed TodX, BenzABCD, and the upper operon, nano dropped,digested, and ran the lower operon gibson assembly in a gel. Inoculated and autoclaved beakers in preparation for making more pre-competent cells as well as the next iteration in the MIC50 assay.

**Vi Ka:** Helped in first part of bacterial swab genomic DNA extraction with Katie. Attempted to prepare inoculation colonies for SDS-PAGE, however it was discovered that the colonies were growing at an abnormally slow rate. Cultured new plates of G-4. Collected Blood Agar plates, as a kind donation from the Lily Media Prep Room. Did miniprep for all lower operons.

## Tuesday, 7/18 Day 49

**Andrew:** Created 30 new LB agar plates for preservation protocol and for other. Created three X-gal/IPTG/amp plates for pGEM transformations. Introduced competition into full scale model for engineered bacteria and for native lung bacteria. Made sure model could handle calculations for a couple days of time with new methods. Plated first day of preservation protocol.

**Archana:** Trying to transfer our domain to Jimdo. Attended euthanasia lab. Helped put lungs in nutrient media, take swabs, and ventilate. Helped use vibratome on lungs.

**Caleigh:** Vibrotome training. Arranged for Pipettmans to be checked. Wrote lab note for Experiment campaign.

**Chris:** Transformed two unsuccessful wells for interlab study.

**Katie:** Cleaned out and set up Sanyo incubator. Observed Bartlett lab rat sacrifice and brain slice with vibratome. Made a solution of GFP-transformed E. coli and PBS buffer. Attended euthanasia lab. Swabbed lungs, lavaged with E. coli solution, ventilated, swabbed again. Attempted to use vibratome to no avail. Put lungs in a 2% agarose gel and attempted to use vibratome again, with no luck. Cleaned lab.

**Kevin:** Prepared more competent cells, transformed something, resuspended promoters

**Vi Ka:** Minipreped upper operon x2, lower operon and GFP. Inoculated g-block 4.

## Wednesday, 7/19 Day 50

**Andrew:** Read CFU counts from previous day. Plated preservation protocol tubes. Continued model work and implemented definite competition with benzene degradation affecting growth rate.

**Archana:** Emailed Dr. Rickus about Experiment Campaign funding. Responded to Cook Biotech contact. Emailed Jimdo support to solve domain issues. Emailed our Purdue iGEM admin contact for access to Jimdo emails. Created list of materials and budget for Imagination Station Strawberry DNA activity. Sent out LinkedIn messages and emails to iGEM alumni for donations.

**Caleigh**: Planned and delegated daily labwork. Prepared an Experiment.com lab note about the lung assay. Contacted alumni and biotechnology companies to encourage donations to our Experiment campaign.

**Chris:** Made gel to run remaining digests (lower operon, GFP) and Kevin’s PCR (G2, G3).

**Katie:** Took half day. Edited and posted Experiment note about lungs. Posted to social media about Experiment campaign. Edited slides for tomorrow’s meeting

**Kevin:** PCR, gel purification, excision, and extraction for G-blocks 1-3. Autoclaved more LB broth. Transformed G4 ligation product on Amp/Xgal plates, etc. Inoculated in preparation for MIC50 assay tomorrow.

**Vi Ka:** Prepared digest for GFP (x2), lower operon. Made gel to run Gibson G4-6, TodX, and BenzABCD - some of them looked like just the backbone. Made another gel in the afternoon to run remaining digests (lower operon, GFP), and Kevin’s PCR (G2, G3)

## Thursday, 7/20 Day 51

**Andrew:** Read CFU counts from previous day. Plated preservation protocol tubes. Added transport from TodX to mathematical model. Attended and presented at weekly lab meeting.

**Archana:** Contacted Purdue iGEM alumnus again to fix website. Emailed back Fischer Scientific about gloves. Contacted Susan about leak. Edited weekly presentation slides. Tried to contact Assoc. Dean of Research and Creative Endeavors for the Honors College. Printed time cards. Emailed ABE business office regarding funding transfer. Posted about Experiment Campaign on social media.

**Caleigh:** Assigned and delegated daily labwork. Prepared slides for our daily lab meeting and attended lab meeting.

**Chris:** Conducted literary research for benzene uptake in lungs and relative benzene degradation due to lung biome metabolism.

**Katie:** Performed Miniprep with Vi Ka on GFP 1 and 2 (248.6, 256.9 ng/ul, respectively). DNeasy prep of lung swabs to see how the pipette tips worked as swabs. Showed Kok Vi Ka’s beautiful miniprep shirt. Wrote thank you notes to Experiment backers. PCR and gel of DNA extraction, which failed miserably.

**Vi Ka:** Performed Miniprep with Katie on GFP 1 and 2 (248.6, 256.9 ng/ul, respectively). Worked on DNeasy prep of swabs from the rat lungs. Showed Kok my beautiful miniprep shirt. Made PCR for bacterial swab, checked gel. No results.

## Friday, 7/21 Day 52

**Andrew:** Read CFU counts from previous day. Plated preservation protocol tubes. Autoclaved Eppendorf tubes and LB broth. Created object-oriented Python script for model that functions with competition, growth, benzene degradation, and enzyme kinetics throughout entire pathway.

**Archana:** Turned in time cards. Emailed Pam regarding Experiment payment transfer. Emailed Experiment about payout method. Contacted companies for funding. Edited week 9 newsletter.

**Caleigh:** Prepared poster for Imagination Station public engagement activity. Cleaned the lab to prepare for REM inspection. Submitted the lower operon for sequencing.

**Chris:** Created fluorescein dilution prep for Monday’s cell measurement plate-reading procedure. Also, prepped for standard calibration curve for readings.

**Katie:** Sick in the morning but came in later when feeling better. Wrote the weekly newsletter and the weekly Biomakers blog. Posted to social media about how much we love IDT. Messaged with Team Peshawar about our Skype session next Wednesday.

**Kevin:** Inoculated upper operon colonies, Prepared the following plates: LB amp, LB chlor, LB amp-chlor, and LB IPTG/xGal/amp.

**Vi Ka:** Prepared pipette tips to autoclave. Did miniprep for four GFP - obtained results between 200ng/uL - 350ng/uL. Prepared it for digestion. Prepared gel and loaded the digestion mixture with Katie and Chris. GFP 1 was very faint; only excised GFP 2, 3, 4, K. T0 and T5 (from previous day’s bacterial swab) had no DNA too.

[Back to Top](#top)

## Week 10

## Monday, 7/24 Day 53

**Andrew:** Read CFU counts from previous day. Plated preservation protocol tubes. Created all-in-one Python script for model that predicts equilibrium benzene concentration, cell equilibrium concentration, time of benzene degradation during equilibrium concentration, and time after benzene has been degraded until equilibrium is reached.

**Archana:** Extended Experiment campaign by a week! Sorted Jamboree registration details. Updated Jamboree Event in Boiler-Link. Called contact for university funding, with no success. Called Sheraton Hotels regarding deposit fees. Updated Jamboree event form.

**Caleigh:** Kept tracking down potential Experiment.com backers. Contacted potential collaborations and human practices connections. Inoculated for Interlab study and hardware testing.

**Chris:** Completed interLab study Day 1 survey. Proposed changing time step to Andrew’s model.

**Katie:** Attended SURF seminar in which she presented a “one minute thesis” about her research. Wrote email to Brandon about getting more lungs. Wrote email to Foundry about getting funds and mentorship on finding our unmet need and target population.

**Kevin:** Part of miniprep for the upper operon, digest of upper operon, gel purification and imaging of upper operon. Made new chlor, amp, and chlor-amp plates and plated one of each with wild type overnight to test the antibiotic.

**Vi Ka:** Called SCU China. Took the day off.

## Tuesday, 7/25 Day 54

**Andrew:** Read CFU counts from previous day. Plated preservation protocol tubes. Incorporated framework for all aspects of the first iteration for the full scale model. Created ability to write data to .txt files which can be copied into Excel to form graphs.

**Archana:**  Attended meeting for follow up with Dr. Fernandez. Emailed Jimdo support. Listed ways iGEM can market SynBioBeta’s conference. Collected inoculation loops from Kok. Sent messages to Experiment backers. Responded to Experiment support to label check with iGEM. Edited Biomakers website.

**Caleigh:** Worked on creating wiki content. Contacted potential human practices connections. Kept trying to get Experiment campaign backers.

**Chris:** OD600 all Interlab study samples then completed cell concentration dilutions for plate reader protocol. With help of Suraj, ran the plate reader to check absorbance and fluorescence overnight every two hours. Created a script to help with protocol.

**Katie:** Worked on presentation for SURF. Attended meeting with Dr. Fernandez. Got inoculation loops from Kok for lung work.

**Kevin:** Gibson PCR check of the atpIp promoter, organized the -20 freezer, digested one lower operon and 5 upper operon out of pgem plasmids, gel purified and imaged, made amp/IPTG/xGal plates. Attended meeting with Dr. Fernandez.

**Vi Ka:** Attended meeting with Dr Fernandez. Filled up pipette tips. Did miniprep for upper operons A-E, and lower operon 2.

## Wednesday, 7/26 Day 55

**Andrew:** Read CFU counts from previous day. Plated preservation protocol tubes. Re-introduced varying time step into mathematical model. Fine-tuned parameters needed in order to leave open framework for editing. Incorporated data from preservation protocol. Ran sensitivity analysis on chosen parameters from model.

**Archana:** Tried to locate Dr. Kristina Bross for funding around campus. Finished uploading all blog posts from past years onto website.

**Caleigh:** Uploaded survey and began distribution. Hardware testing.

**Chris:** Explored Cotter’s Method with Andrew to determine most sensitive parameters on system (optimization). Disappointed that plate reader overwrote data collected over 6 hour duration.

**Katie:** Came in late as 1/2 vacation day. Worked on slides for SURF presentation.

**Kevin:** Transformed Upper Operon, Lower Operon, and G4. MIC50 iteration

**Vi Ka:** Did miniprep for GFP, and five Upper Operons. Ran gel for all pre-digestion, miniprepped products; discovered that there was only genomic DNA present and no plasmid for the upper operons. Proceeded with GFP digestion.

## Thursday, 7/27 Day 56

**Andrew:** Completed presentation on mathematical modeling to organize thoughts, present data, and show direction. Modified steps taken in model for growth in order to allow for constant benzene uptake from lung and from bacteria. Presented modeling work along with preservation protocol data at weekly lab meeting. Read CFU counts from previous day. Plated preservation protocol tubes.

**Archana:** Uploaded latest blog posts, advisor bios, and student bios. Edited presentation slides.

**Caleigh:** Created presentation slides and attended presentation. Analyzed hardware testing results. Began documenting data from survey.

**Chris:** Went to presentation :)

**Katie:** Listened to Andrew sing and talk about modeling forever. Practiced presentation. Helped Vi Ka in wet lab.

**Kevin:** Inoculated colonies from original lower operon (one of which had the correct insert), and the newly transformed upper and lower operons.

**Vi Ka:** Miniprepped GFP, digest, and gex extracted. No bands whatsoever - suspect something went wrong with the gel. Overheated it in the microwave?

## Friday, 7/28 Day 57

**Andrew:** Created 18 new LB agar plates (small). Read CFU counts from previous day. Plated preservation protocol tubes. Continued research on relation between biomass and growth rate along with glucose/benzene uptake. Held meeting with modeling team to discuss final goals before summer ends.

**Archana:** Updated student bios on website. Gave feedback to Katie on SURF presentation. Responded to MSU iGEM. Emailed Kristina Bross for funding from Honors College. Edited week 10 Newsletter.

**Caleigh:** Assigned daily labwork. Analyzed survey results. Began preparing an Experiment note about iGEM.

**Chris:** Attempted calibration curve using plate reader for Interlab study. Planned out new trials for interlab study - (Monday calibration curve, Tuesday bacterium)

**Katie:** Helped Vi Ka in miniprepping 13 tubes. Practiced SURF presentation. Wrote weekly newsletter.

**Kevin:** Researched possible improvements to be made to the MIC50 assay. Inoculated RFP and added more LB to all miniprepped reactions. Inoculated GFP for future use with the universal promoters and RBS’

**Vi Ka:** Miniprepped 13 tubes. Took a half day off.

[Back to Top](#top)

## Week 11

## Monday, 7/31 Day 58

**Andrew:** Read positive on positive control and negative on negative control. Continued research on benzene transport rates. Autoclaved new LB broth and nanopure water. Continued research on constraints for flux balance analysis and competition coefficient calculations for mathematical model.

**Archana:** Submitted club dues and talked to SOGA about approving Jamboree event. Edited Jamboree lab note. Uploaded annual competition updates onto website. Attended Purdue Foundry meeting with Juliana Casavan.

**Caleigh:** Planned and delegated daily labwork. Analyzed survey data. Created wiki content.

**Chris:** Well-plated Interlab study LUDOX and standard curve solutions

**Kevin:** Ran colony PCR gel: no results for upper operon, genomic DNA for lower operon, and a successful band for atpIp. Performed GFP miniprep, retransformed the upper operon on one of our chlor plates and one of Kok’s plates, inoculated the lower operon (the old 5 colonies), the successful atpIp, and more GFP/RPF/wild type E. coli, colony PCR for the lower operon again, gel excision and extraction for GFP

**Vi Ka:** Did miniprep. Took a half day.

## Tuesday, 8/1 Day 59

**Archana:** Edited budget and updated reimbursements. Emailed Kristina Bross from Honors College regarding transfer of funds. Showed Katie how to upload piktochart. Found that PNG files have higher resolution. Edited Summer Update 10 for the blog. Began registering team for Jamboree.

**Caleigh:** Analyzed survey data. Created wiki content. Miniprepped and digested the lower operon. Ran a gel with digests and PCR products.

**Chris:** Gathered data for InterLab Standard Curve and Calibration Protocol. Started Piktochart for InterLab study webpage.

**Kevin:** Made 40 tubes of competent cells and tried the MIC50 assay again (was relatively successful).

**Vi Ka:** Did miniprep for GFP 1, 2, 3. Took a half day.

## Wednesday, 8/2 Day 60

**Archana:** Emailed Experiment about payout method. Helped Katie swab, ventilate, and fill lungs with agarose. Contacted Experiment about deadline extension. Contacted former iGEM members who might be willing to donate. Registered team members for Jamboree. Updated budget.

**Caleigh:** Analyzed survey data. Created wiki content. Helped with using vibratome on lungs. Miniprepped and digested GFP. Inoculated for hardware. Transformed universal RBS and promoters.

**Chris:** Diluted cell concentrations for Interlab study. Created plate reader script to read data every 2 hours overnight.

**Kevin:** Gibson for atpIp long and RBS 1-3. PCR check for the upper operon Gibson products, colony PCR for the two colonies on the upper operon plate and for 5 different colonies on the lower operon plate (one of the upper operons was successful). Gel purification and extraction of GFP. Inoculation of lower operon colonies 6-10 and upper operon colony 2, replated lower operon colonies 1-5 to increase chance of successful colony PCR and also plated E. coli on a newly made chlor plate to test its viability.

## Thursday, 8/3 Day 61

**Archana:** Emailed all news agencies with press release. Responded to Purdue Exponent. Worked on iGEM history piktochart. Updated weekly presentation meeting slides. Researched Foundry contact.

**Chris:** Collected data from plate reader. Created visual displays for data. Created wiki content.

**Katie:** Wowed the audience with her SURF presentation.

**Vi Ka:** Left on a secret (sweet) mission to get us more funding.

## Friday, 8/4 Day 62

**Archana:** Turned in time cards and packaging slips to Pam. Requested account statement. Responded to Purdue Exponent and gathered photos for our spread. Requested wire transfer for Jamboree attendance fee. Finished uploading all lab notebook entries. Spoke to Exponent for news spread. Had meeting with Foundry. Compiled list of members/info for Exponent and pitch for Foundry.

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)


[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:Purdue/Experiments

Toggle navigation

[![Purdue](https://static.igem.org/mediawiki/2017/d/da/Purdue_Logo.png)](https://2017.igem.org/Team%3APurdue)

* Team
  + [Team History](https://2017.igem.org/Team%3APurdue/Team)
  + [Meet the Team](https://2017.igem.org/Team%3APurdue/MeetTheTeam)
  + [Team Collaborations](https://2017.igem.org/Team%3APurdue/Collaborations)
* Project
  + [Description](https://2017.igem.org/Team%3APurdue/Description)
  + [Problem](https://2017.igem.org/Team%3APurdue/Problem)
  + [Pathway Design](https://2017.igem.org/Team%3APurdue/Design)
  + [Lung Microbiome](https://2017.igem.org/Team%3APurdue/lungmicrobiome)
  + [Genetic Construct Design](https://2017.igem.org/Team%3APurdue/GeneticDesign)
  + [Experiments and Results](https://2017.igem.org/Team%3APurdue/Experiments)
  + [Notebook](https://2017.igem.org/Team%3APurdue/Notebook)
  + [InterLab](https://2017.igem.org/Team%3APurdue/InterLab)
  + [Model](https://2017.igem.org/Team%3APurdue/Model)
  + [Demonstrate](https://2017.igem.org/Team%3APurdue/Demonstrate)
  + [Attributions](https://2017.igem.org/Team%3APurdue/Attributions)
* Parts
  + [Parts](https://2017.igem.org/Team%3APurdue/Parts)
  + [Basic Parts](https://2017.igem.org/Team%3APurdue/Basic_Part)
  + [Composite Parts](https://2017.igem.org/Team%3APurdue/Composite_Part)
* [Safety](https://2017.igem.org/Team%3APurdue/Safety)
* Human Practices
  + [Silver HP](https://2017.igem.org/Team%3APurdue/HP/Silver)
  + [Integrated and Gold](https://2017.igem.org/Team%3APurdue/HP/Gold_Integrated)
  + [Public Engagement](https://2017.igem.org/Team%3APurdue/Engagement)
* Awards
  + [Applied Design](https://2017.igem.org/Team%3APurdue/Applied_Design)
  + [Entrepreneurship](https://2017.igem.org/Team%3APurdue/Entrepreneurship)
  + [Hardware](https://2017.igem.org/Team%3APurdue/Hardware)
  + [Model](https://2017.igem.org/Team%3APurdue/Model)
* [Judging Form](https://igem.org/2017_Judging_Form?team=Purdue)

![](https://static.igem.org/mediawiki/2017/1/12/Purdue-experiments-home.png)

[![](https://static.igem.org/mediawiki/2017/0/04/Upromoter-icon_25769815.png)](https://2017.igem.org/Team%3APurdue/UniversalPromoter)

[![](https://static.igem.org/mediawiki/2017/4/42/Purdue-lung-mimic-icon.png)](https://2017.igem.org/Team%3APurdue/Experiments/lungmimic)

[![](https://static.igem.org/mediawiki/2017/7/74/Preservation_outline.png)](https://2017.igem.org/Team%3APurdue/Experiments/BacterialPreservation)

[![](https://static.igem.org/mediawiki/2017/c/c0/Purdue-preservation-button.png)](https://2017.igem.org/Team%3APurdue/Experiments/Benzene_Operon)

[![](https://static.igem.org/mediawiki/2017/2/26/Universalrbsicon.png)](https://2017.igem.org/Team%3APurdue/Experiments/RBS)

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:Purdue/Results

Toggle navigation

[![Purdue](https://static.igem.org/mediawiki/2017/d/da/Purdue_Logo.png)](https://2017.igem.org/Team%3APurdue)

* Team
  + [Team History](https://2017.igem.org/Team%3APurdue/Team)
  + [Meet the Team](https://2017.igem.org/Team%3APurdue/MeetTheTeam)
  + [Team Collaborations](https://2017.igem.org/Team%3APurdue/Collaborations)
* Project
  + [Description](https://2017.igem.org/Team%3APurdue/Description)
  + [Problem](https://2017.igem.org/Team%3APurdue/Problem)
  + [Pathway Design](https://2017.igem.org/Team%3APurdue/Design)
  + [Lung Microbiome](https://2017.igem.org/Team%3APurdue/lungmicrobiome)
  + [Genetic Construct Design](https://2017.igem.org/Team%3APurdue/GeneticDesign)
  + [Experiments and Results](https://2017.igem.org/Team%3APurdue/Experiments)
  + [Notebook](https://2017.igem.org/Team%3APurdue/Notebook)
  + [InterLab](https://2017.igem.org/Team%3APurdue/InterLab)
  + [Model](https://2017.igem.org/Team%3APurdue/Model)
  + [Demonstrate](https://2017.igem.org/Team%3APurdue/Demonstrate)
  + [Attributions](https://2017.igem.org/Team%3APurdue/Attributions)
* Parts
  + [Parts](https://2017.igem.org/Team%3APurdue/Parts)
  + [Basic Parts](https://2017.igem.org/Team%3APurdue/Basic_Part)
  + [Composite Parts](https://2017.igem.org/Team%3APurdue/Composite_Part)
* [Safety](https://2017.igem.org/Team%3APurdue/Safety)
* Human Practices
  + [Silver HP](https://2017.igem.org/Team%3APurdue/HP/Silver)
  + [Integrated and Gold](https://2017.igem.org/Team%3APurdue/HP/Gold_Integrated)
  + [Public Engagement](https://2017.igem.org/Team%3APurdue/Engagement)
* Awards
  + [Applied Design](https://2017.igem.org/Team%3APurdue/Applied_Design)
  + [Entrepreneurship](https://2017.igem.org/Team%3APurdue/Entrepreneurship)
  + [Hardware](https://2017.igem.org/Team%3APurdue/Hardware)
  + [Model](https://2017.igem.org/Team%3APurdue/Model)
* [Judging Form](https://igem.org/2017_Judging_Form?team=Purdue)

This page is under construction. Please come back soon!

# Results

Here you can describe the results of your project and your future plans.

##### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

##### You should also describe what your results mean:

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember all measurement and characterization data must be on part pages in the Registry.
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

##### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

##### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)