Posted

Week of June 12 – June 16

 

06.12.2017

Monday was a great way to start off the week as we found that our Experiment campaign had already received two donations, we received a lifetime supply of pipette tips from VWR, and we found that our cells were viable under all tested conditions after two days of preservation! We started on a BnzABCD in-vitro enzyme activity assay and prepared for our first full run of bacterial preservation; we also worked on Gibson and pGEM transformations. In the dry lab, we modified the universal promoter to make the ratios of constitutive organisms more desireable and sent an email to an employee at the BP Whiting plant, in the hopes that we can set up a Human Practices meeting. Keep your fingers crossed!

 

06.13.2017

Tuesday was a busy day for the wet lab! We performed periodic viability checks for our bacterial preservation assay to determine the upper limit of storage within each medium, ran a PCR amplification of all gBlocks along with a GFP digest to test universal RBS strengths and ran the digest through a gel, performed gel purification, tested dilution factors for colony forming unit (CFU) counts, and began pGEM on gBlocks 1-3. We also researched the effects of introducing bacterial species native to the lung microbiome to different human microbiomes and completed the design of the universal promoters.

 

06.14.2017

We met with Dr. Amber Hopf Jannasch, the lab manager for BIND 134 on Wednesday morning to get the ins and outs of her lab supplies and safety training. In the wet lab, we performed pGEM and Gibson transformations, gel extraction on GFP-transformed bacteria and gBlock 6 PCR product, and touchdown PCR on gBlocks 4-6 with DMSO, glycerol, or GC enhancer as additives to troubleshoot our PCR struggles; none of them worked. We also tested the viability of bacteria suspended in a variety of mediums and stored in different preservation conditions the previous day. The dry lab work was more productive; we are learning how to use Bootstrap on websites to make our wiki look more clean and modern and we emailed potential Human Practices contacts, including the Indiana State Department of Health Tobacco Use Prevention and Cessation Commission, about meeting to discuss their feelings about our product. We also reached out to the University of Michigan about a potential collaboration and summer meeting!

 

06.15.2017

On Thursday, two team members met with PhD candidate Hakeem Abdul Wahab from the statistics department to get feedback on our Human Practices survey and discuss how we want to draw conclusions from the data we get once our survey is distributed. In the meantime, we repeated PCR for gBlock 4 (for the final time!) and inoculated the white colonies from Wednesday’s pGEM transformation. Our test run of the preservation assay ended, telling us that E. coli can be stored in nanopure water, sucrose, or sucrose and milk at -80 and -20°C temperatures for at least five days and still produce viable cultures. The full, two-week run will begin tomorrow. We also finished designing the universal promoter, and we plan to order it soon! Finally, we had a PI meeting with Drs. Rickus and Solomon to receive feedback on our project; so far the project is on track, and our advisors are very pleased with our progress!

 

06.16.2017

We ran our first iteration of the preservation protocol on Friday. Our first dilution tests were plated after an hour of freezing for each condition: -80 or -20 °C and nanopure water or milk and sucrose media. Additionally, we continued our pGEM transformations by performing ligation and digestion on the pGEM plasmid for gBlocks 1-3.

 

Weekend

On Saturday and Sunday, our dedicated team members returned to the lab to verify dilution factors and plate the preservation protocols from Friday. We also performed a miniprep on gBlock 3 in the pGEM T-easy vector, as well as digested and gel purified GFP from a kit plate. Finally, we performed a bacterial transformation of gBlocks 4-6 pGEM ligations.

 

E. Coli with G1 pGEM transformation plate

pGEM ligation results: While there are many blue colonies, a few white colonies (meaning a successful transformation) were identified (marked with black Sharpie dots).

Pie chart of Universal Promoter genera contribution to design.

Chart of Universal Promoter Genera Distribution: This pie chart shows how the distribution of genera which contributed to the design Universal Promoter developed over each iteration. The final iteration is shown in the middle and the genera contribution percentages are labeled. In theory, each of these genera should be able to express the promoter.

Viability assay plating with a variety of colonies; each colony was plated after a certain number of hours to prove that the culture was viable after being in a freezer for an extended period of time.

Viability Tests of Preservation Assay: The preliminary assay to determine cell viability in a variety of temperatures and media over a week showed that the cells were able to survive and be cultured. A more in-depth assay will be performed this week.

Posted

In an effort to raise funds for laboratory supplies, this year the Purdue iGEM team is launching another crowdfunding campaign on the Experiment website, a platform similar to GoFundMe and Kickstarter but exclusive to scientific endeavors. The fundraising goal is ambitiously set at $4,000, and the team would greatly appreciate any and all help it can get. There is a short project description on the Experiment page in addition to a breakdown of how contributions will be invested, also featured below. Thank you for supporting our team by donating to and sharing the page!

Posted

Week of June 5 – 10

06.05.2017

Monday continued the wet lab work from the weekend, as we repeated PCR for gBlock 4 with Q5 Polymerase. We also updated the metabolic model with an E. coli core model and continued to modify protocols. Additionally, we updated the Human Practices contact list to include more potential experts and companies, including BP’s Whiting Refinery. We attempted to contact them to schedule a meeting, with no luck.

06.06.2017

On Tuesday, we ran a gel on the Q5 PCR products, excised the correct bands, and performed gel purification; as gBlock 4 PCR failed again, we revised the annealing temperature of the reaction and tried again. We continued reviewing and updating protocols and continued working on the metabolic modeling script.

06.07.2017

Wednesday, we revised the PCR protocol again for gBlock 4 and performed a Gibson assembly on gBlocks 1-3. More protocols were edited and we contacted fellow Bindley researcher Adrian Ortiz to help us with the metabolic modeling.

06.08.2017

On Thursday, we booked our flights and hotels for the Giant Jamboree and had fun discussing our plans for Boston! We ran the modeling script to optimize objective functions. In the wet lab, we began testing bacterial preservation with milk powder and performed a transformation with the product of the Gibson assembly. Additionally, we drafted emails to send to Human Practices contacts.

06.09.2017

Friday was very exciting in that we found out that our preliminary bacterial preservation assay worked! We also performed PCR on the singular colony that formed from Thursday’s Gibson transformation. We also edited the Experiment campaign and press release documents. Our Experiment Campaign starts this weekend!!

06.10.2017

Over the weekend, more dedicated team members made plates for the bacterial preservation test run conditions and started a preservation test run using sucrose as the medium for the cell suspension. Read more »

Posted

Week of May 29 – June 4

05.29.2017

Monday was Memorial Day, so we enjoyed a long, relaxing weekend!

05.30.2017

Tuesday, however, we got right back to work! The universal ribosome binding sites arrived from IDT. We began reaching out to experts for our Human Practices work. Work was started on metabolic pathway modeling and we found five potential universal promoter sequences! Additionally, we began looking into the lung mimic logistics; we contacted Drs. Breur and Kasinski about donating rat lungs from their laboratories to the project and made a spreadsheet of common genera of bacteria in the lung microbiome and the optimal culture conditions for each. At the end of the day, we performed a GFP transformation on E. coli to practice the protocol.

05.31.2017

On Wednesday, we had our first Human Practices meeting with Dr. Umberger, the Purdue Polytechnic Associate Dean for Engagement who teaches a class on the FDA regulatory procedures. She discussed with us some considerations we need to make in developing our product, including the unmet need our product would fill and concerns about the product being introduced to other human microbiomes (i.e. the skin and eyes). She also generously offered to introduce us to a contact at Cook Biotech that might be interested in sponsoring our team! Our Experiment campaign was submitted for review; more details on that once it is live! We continued writing up protocols for future assays and working on a code to model the enzyme kinetics and reaction rates.

06.01.2017

On Thursday, we did more wet lab work! We performed the miniprep protocol on the GFP-expressing E. coli. In continuing the work from previous days, we researched and wrote up protocols for analyzing enzyme activity and transport assays. We also looked into the populations which are most often exposed to dangerous levels of benzene in order to determine our initial target population. Our project’s modeling script produced graphs for our data, but we were advised during our weekly PI meeting to consider flux balance analysis as a way to model bottlenecks in the pathway. Also at the meeting, we decided to use mammal lung decellularization and recellularization as our lung mimicking method! We are very excited to revive our own lungs in the coming weeks!

06.02.2017

Friday, we continued improving upon the modeling code to incorporate changing concentrations and flux balance analysis. We reached out to companies regarding funding, mentorship, discounts, and in-kind donations. In the wet lab, we created a master mix of the gBlock DNA and the primers, poured agar plates, and performed PCR on the six gBlocks we have.

Weekend

Over the weekend, some team members were dedicated enough to come in and perform a quick gel check on the PCR products. We determined that the results were not as expected, so the next day, the team came back and redid PCR on the six gBlocks with Q5 buffer and performed a gel check. All but gBlock 4 had the expected bands.

WebLogo Plot of universal promoter sequences.

Universal Promoter WebLogo Plot: This WebLogo plot, made with Berkeley’s WebLogo application, shows the frequency of amino acids to create sequences of potential universal promoters. Five of the sequences were chosen based on how conserved their -35 to -10 regions are.

 

Graph of Concentration (mM) vs Time (s)

Concentration vs. Time Metabolic Modeling Graph: This is the first graph that was created with our metabolic modeling script. More factors are needed to accurately model the system, but the script is off to a great start!

Posted

Week of May 22, 2017 – May 26, 2017

05.22.2017

The first day began with a tour of our new home for the summer, the Bindley Bioscience Center. Susan, our favorite administrative assistant, showed us around and gave us the basic safety training. The remaining hours of the day were spent designing universal promoters and ribosome binding sites, creating protocols for future assays, identifying methods for a lung mimic creation, and reviewing literature for identifying bacteria living in the lungs. We resuspended the DNA primers that we designed at the end of the day.

05.23.2017

On Tuesday, we enjoyed writing our biographies for the Experiment campaign and the Wiki before continuing the work that we had started the previous day. We also contacted researchers studying bacteria in the lungs, asking for culture donations.

05.24.2017

On Wednesday, we had our first team meeting with our advisor Dr. Rickus in which we presented the work of the week. We got feedback about our progress so far and experts we should contact for advice. We found five possible universal RBSs (see image below), all of which are novel to the iGEM registry, and ran them through a software that models transcription rates. A bill of materials was started and we contacted companies about discounts and in-kind donations. We also received an email from Dr. Weimer of the USDA, offering to donate Megasphaera elsdenii culture!

05.25.2017

Thursday, we prepared for a meeting with our other advisor, Dr. Solomon, and graduate student advisors. At this meeting, he gave us more feedback about our work thus far and recommended other possible on-campus contacts. We wrote up more protocols for creating gene constructs, preserving bacteria, transport assays, benzene quantification, and in-vitro enzyme activity.

05.26.2017

The beginning of Friday included our team meeting with Dr. Sors in which we discussed our project and he gave us some ideas for expert on-campus contacts who would be willing to help us with our project. Afterward, Kevin, Caleigh, and Archana met with Dr. Cooper from the Bindley Metabolite Profiling Center and received approval to use his gas chromatograph for our benzene, toluene, and xylene quantification protocol! Additionally, we decided upon a method for preserving bacterial cultures for long-term storage, created gBlocks from the universal ribosome binding sites, and completed more protocol writeups.

 

Logo plot of compared ribosome binding sites

Universal Ribosome Binding Site WebLogo Plot: The above image shows potential universal ribosome binding sites found by comparing various RBSs from species of bacteria known to inhabit the lungs and throat, as well as other unrelated bacteria. Five sequences found are novel to the iGEM registry. This image was created with Berkeley’s WebLogo application.

 

Posted

It’s summer again, meaning that it is time to introduce the new Biomakers iGEM project! Keep up with the team’s progress with weekly updates on this website!

Animated Lung Graphic with bacteria shown

Take a big, deep breath in. Now breathe out. Filling your lungs with clean air is so relaxing, right? You might take a deep breath before taking a big test, while meditating, or at the end of a long day at work. However, some people live and work in environments where taking a breath, let alone a deep one, is unhealthy, exposing them to toxins in the air. One of these toxins is benzene, a carcinogen found in cigarette smoke, automobile exhaust, and industrial pollution, among other things. Gas station attendants, factory workers, and people living in inner cities are a few examples of populations that breathe this molecule in during their everyday lives and thus are more susceptible to developing lung diseases and cancer.

The 2017 Purdue Biomakers are working to develop probiotics to degrade benzene buildup in the lungs, preventing adverse effects of the toxin. The team is engineering the native bacterial species of the lung microbiome to break down benzene into pyruvate and acetyl-CoA, molecules used in the cell’s natural metabolism. Additionally, the Biomakers are designing a drug-delivery device, similar to an inhaler used to deliver asthma medication, in order to introduce our product to the lungs. The team hopes that the product will be used by those who are occupationally exposed to benzene, as well as smokers who are trying to quit.

Image courtesy of Cystic Fibrosis News Today.

Posted

11 weeks. 77 days. 1,848 hours. 110,880 minutes. 6,652,800 seconds. While not all spent in lab, these numbers represent the time of a summer spent learning, exploring, and discovering. At the conclusion of this week, the team’s full-time work in Bindley Bioscience Center will conclude as class begins after a brief respite. While there is still much to do, much has already been done. Now, the team is looking forward to the school year’s accomplishments as time continues ticking toward the Giant Jamboree in Boston at the end of October! A heartfelt thank-you to everyone who has supported this endeavor; it would not have been successful without you.

Summary of Lab Work

1. Development of theoretical genetic constructs for both phosphorus uptake and organic nanowire components of the project.

2. PCR amplification, digestion, ligation, gel electrophoresis, and transformation of individual M. phosphovorus genes into E. coli. Currently testing for functional characterization and protein expression.

3. In collaboration with the USDA National Soil Erosion Research Laboratory, construction of a prototype bioreactor from 5-gallon buckets, tubing, water filters, and an aquarium pump with complementary MATLAB model and silica beads for bioencapsulation.

4. Participation in InterLab study to standardize fluorescence units.

5. Collaboration discussions and activities with Exeter, Northwestern, UNL, UChicago, SVCE, and Rose-Hulman iGEM teams.

6. Human practices and outreach including fully-funded Experiment campaign, award of SYNENERGENE grant, a booth at the Wabash River Fest, media coverage, and a survey assessing knowledge of wastewater treatment practices that you can explore here if you haven’t already.

lab bench

Image courtesy of mediabakery.com

Posted

Monday, July 25

Preliminary trials of testing for phosphorus using the lachat and the ICP-OES at the USDA National Soil Erosion Research Laboratory began. Both machines operate via spectrophotometry, comparing measured values to standard curves. The lachat measures orthophosphate, or soluble reactive phosphorus, while the ICP measures total phosphorus, the entirety of phosphorus contained in a sample. Corresponding with this endeavor, many bottles of Tris-HCl buffer, the media in which phosphorus-eating E. coli will be suspended, were created and autoclaved for future use.

Tuesday, July 26

The week continued by learning to make methanol-free bioencapsulation beads using the sol-gel method. A graduate student in the lab worked with team members to describe and demonstrate the protocol. After practice forming the beads, the team switched to a different method that could be more easily controlled and manipulated. E. coli were added to the beads to determine whether or not they would be able to survive within the structure.

Fluorescent Beads 1

Above: Shots of silica beads containing E. coli expressing green fluorescent protein, GFP, (left) and red fluorescent protein, RFP, (right) imaged under EVOS fluorescence microscope; the circled areas on the left indicate particularly bright spots where it can be reasonably inferred that a clump of E. coli is successfully producing GFP

Silica Beads 2

Above: Additional images of silica beads with E. coli expressing RFP (left) and GFP (right); the brightly-glowing RFP beads indicate RFP expression

Wednesday, July 27

Based on results from the lachat and ICP and the discovery that E. coli would not grow in the originally proposed Tris-HCl buffered solution, the measurement of phosphorus uptake protocol was modified after a productive meeting with advising scientists. Modified microbes will now be suspended in a minimal media solution whose components can be processed by both the sensitive lachat and ICP instruments. Prototype modeling progressed, with team member Barrett composing a 3D model to better-depict uptake system hardware. In the afternoon, a few team members also visited the West Lafayette Wastewater Treatment facility, where they learned about the local area’s methods for phosphorus removal featuring their newly-implemented chemical system.

3D Bioreactor

Above: Current bioreactor prototype with 2 5-gallon buckets, an aquarium pump, connective tubing, and 3 standard water filter chambers to house E. coli

Thursday, July 28

Additional trials of media and various water samples were run on both the lachat and ICP as team members were trained in their use. Colony PCR and sequencing to confirm the transformation of phosphorus genes into E. coli were also begun, with completion planned to coincide with the conclusion of next week.

Friday, July 29

Focus was placed on brainstorming possible development strategies of silica beads. Two components combine to make these beads: a sol phase and a buffer (Tris-HCl). The team has been trying to find the proper ratio of these two ingredients so beads solidify at an appropriate rate–too slowly, and an amorphous silica blob forms in the bottom of the mineral oil into which the beads drop; too quickly, and the mixture hardens inside the tubing through which it is dispensed. Cool temperatures slow the reaction, so the team has also tried completing the process in the cold room with mineral oil warmed on a hot plate, but to no avail. The current method involves the use of syringes to inject the two components into a small Y-shaped tube where they mix and then pass through a needle at the bottom to drop into mineral oil. According to calculations, 99,000 beads will be needed to fill a standard water filter canister; that’s a lot of drips!

Posted

Monday, July 18

In collaboration with Exeter, the team will be growing up last summer’s killswitch genetic construct under both aerobic and anaerobic conditions. To begin the week, they set up Dr. Rickus’s anaerobic chamber, which is a large, clear box with thick black gloves attached to reach into it–very mad science-y (example below). This week, culture growth will begin after air tanks to sustain the anaerobic culture arrive.

Anaerobic Growth Chamber

Example of an anaerobic chamber; image courtesy of midlandtech.edu.

Tuesday, July 19

Mid-week discussion centered around using the sol-gel method to suspend E. coli in silica beads with which to fill containers external to the main containment area of the bioreactor. The bacteria will be immobilized within the matrix of silica fibers while phosphorus is still able to pass through, making this solution ideal for easy input and retrieval of E. coli from the bioreactor system. See the diagram below for an explanation of the method.

sol-gel

Infographic depicting the sol-gel procedure to form aerogel; image courtesy of centexbel.be

Wednesday, July 20

Wednesday’s focus was the development of a protocol for the quantification of phosphorus both intracellularly and extracellularly. It involved the creation of a guide for phosphorus on the Experiment page, so if you want to become familiar with the differences between total phosphorus, orthophosphate, and polyphosphate, feel free to check it out.

Thursday, July 21

The lab hosted Peter Oladipupo, a Mandela Washington Fellow, answering questions regarding the project as it relates to the generation of energy. It was enjoyable to hear his perspective and show him the progress being made. The day also brought continued research into the development of a model to both inform experimental decisions and make future predictions.

Friday, July 22

During the week, the team found out that IDT was unable to synthesize another nanowire gene, cancelling the order and decisively tabling the wet lab work for the nanowire side of the project for this competition cycle. So, full steam ahead with phosphorus. Nanowires will be revisited during the school year–strong side project potential!

Posted

Monday, July 11

After a successful weekend participating in the Wabash River Fest, the team jumped back into lab work, plating a new culture of Shewie, miniprepping a promoter for use with digestion the next day, and preparing glycerol stock cultures of successfully transformed parts. A bioreactor brainstorming session with USDA-NSERL staff Dr. Ashley Hammac, Stan Livingston, and Scott McAfee left team members enthused about the prototype to come later in the week. Additionally, the team met with representatives of the Purdue Foundry regarding the possibility of developing a business model for the phosphorus removal system. If lab work continues to go well, this may be something to pursue in the fall.

Bioreactor Sketch

Above: A preliminary sketch of proposed bioreactor design

Tuesday, July 12

Wet lab work consisted of the digestion and ligation of all three remaining phosphorus genes in preparation for transformations into E. coli. Mental effort for the day was directed toward understanding E. coli‘s Pho regulon, the system of genes that control phosphorus within the organism, in an effort to determine which specific sequences of DNA may be helpful to up-regulate in the synthetic microbes to greater improve their efficiency.

Wednesday, July 13

The bioreactor prototype was complete! Many thanks to the staff of the NSERL (National Soil Erosion Research Laboratory) for making it possible.

Bioreactor Prototype

Above: Bioreactor prototype, iteration 1

The prototype consists of two 5-gallon buckets, an aquarium pump and outflow tube to maintain constant water level and flow, and three separate ports around the base to channel water from the main reservoir into water filters that will eventually contain modified E. coli cartridges. Phosphorus is to be collected in these chambers, and effluent is controlled by the valves at the point where water flows from the filters. Further testing of the prototype will include the determination of the proper substrate to contain E. coli, optimum temperature and pH, and best water flow rate.

Thursday, July 14

Thursday morning began with celebration as all of the previous day’s transformations worked with multiple colonies appearing on every plate. Go, team! With all phosphorus genes successfully transformed, the team can now move forward with characterizing proteins. Along with this, some strains will be sent to other collaborating teams for characterization to confirm results.

Friday, July 15

Are synthetic biology terms sometimes confusing? Is scientific jargon difficult to understand? Look for explanation no further than the team’s newly-released glossary of synthetic biology and iGEM terms hosted on the Experiment page.

I'm Cultured

Does this joke make sense? If not, please check out the glossary!

With the new bioreactor prototype, several protocols needed to be written including a standard for measuring water flow and a process for system maintenance. The plan for the upcoming week is to characterize individual proteins and begin conducting tests to determine the optimal substrate for E. coli to inhabit in the bioreactor’s external chambers. Any thoughts on what to name the bioreactor? Tweet @PurdueBiomakers to let the team know what you think.