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Monday, June 6

Monday morning got rolling with the attendance of ABE PhD student Dr. Andi Hodaj’s thesis defense regarding two-stage ditches as a tool to reduce nutrient loads. A two-stage ditch is a variation of a traditional agricultural ditch in which the banks are dredged to create floodplains, dissipating the energy of high-flow water and increasing interaction time between vegetation and water. Purdue’s two-stage ditch utilized in Dr. Hodaj’s was established in 2012 and drains 660 acres; it may be a possible implementation point for the project’s biological method.

two stage ditch enlargement

Above: An illustration of a two-stage ditch from thewhiteriveralliance.org

Additionally, primers arrived, and the Experiment project write-ups were finalized and proofed.

Tuesday, June 7

The Experiment campaign went live! Thank you to both Drs. Rickus and Solomon for endorsing the project, and to those who are project backers–your donations are greatly appreciated. See last week’s previous post for more information; the Experiment page can be found here. Work continued on both the spring publication and protein profiles for the wiki.

Wednesday, June 8

Formal lab meeting #1 was a success with the most highly prioritized goal for the upcoming week to find or generate relevant numbers for the project including necessary flow rates for a microbial fuel cell, an appropriate density of E. coli within it, etc. The rest of the afternoon was spent working on action items from the meeting and printing the spring publication, which is FINISHED.

Thursday, June 9

Team members Barrett Davis, Paige Rudin, and Suraj Mohan attended the Regional Clean Energy Innovation Forum held at Ross-Ade Stadium. Leading scientists from across the country gathered to discuss topics like energy storage, biomass and synthetic biology applications (a conversation that piqued the students’ interest), materials manufacturing, and more. The theme the team took away from the day is best summarized with the words of Purdue President Mitch Daniels during his opening remarks: “It’s only an innovation when it’s useful to someone.” How can new technology of nanowires and microbial fuel cells be integrated efficiently into established systems? Is it a cost-effective solution? Only time will tell, but the team is excited to find out.

Biomakers at Clean Energy Innovation Forum

Above: Barrett, Paige, and Suraj (left to right) pose for a picture at the 2016 Regional Clean Energy Innovation Forum

Friday, June 10

Happy birthday wishes to Barrett, as he celebrated his 20th birthday in the lab on Friday. In the best interest of keeping a sterile work environment, the team refrained from strewing confetti across the bench, although it would’ve been both fun and festive.

Dow AgroSciences generously donated various lab supplies to the iGEM workspace that the team was thrilled to pick up in Indianapolis on Friday morning. Thank you to Dr. Steve Evans, Mr. Mike Doody, and DAS administration for their support in realizing the dream of a self-sufficient, independent lab for the sole use of undergraduate iGEM students. With equipment, it is possible to operate without reliance upon the lab space of other Bindley Bioscience Center scientists, lending additional flexibility to the projects iGEMmers are able to complete.

Phosphorus genes arrived, and the team got to work on PCR amplification in the afternoon. This included resuspending IDT DNA gBlocks and using PCR to increase the amount of DNA present for transformation.

The Weekend

So diligent is this year’s team that several members completed additional PCR amplification and iGEM kit plate promoter transformations on Saturday and Sunday. After digestion and ligation, they did some gel electrophoresis, as well, running several different samples.

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In an effort to raise funds for laboratory supplies, this year the Purdue iGEM team is launching a 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 $3,000, and the team would greatly appreciate any and all the 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 your support!
Experiment campaign budget breakdownWe-want-you-image

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Monday, May 30

Memorial Day was an optional day on the job, but the dedicated iGEM team put in an afternoon of work in Bindley. Practice transformations were successful–both GFP and RFP were apparently expressed, and growth on control plates was as expected. Practice minipreps were next on the agenda, which the interns also completed successfully in addition to some supply inventory.

Tuesday, May 31

Lists of supplies needed for various assays were assembled, and an assessment of necessary ordering was made, thus finalizing the functional assays to run for both nanowire and phosphorus genes. Additionally, brainstorming for an Experiment (crowd-funding for scientific research) campaign was completed.

Wednesday, June 1

Background research continued, and some ideas were tossed around concerning the official project theme for this year (more to come on that later). A meeting with Drs. Rickus and Solomon led to recommendations to practice with both the transmission electron microscope (TEM) and the process of electroporation for genome integration to prepare for performing these procedures later. Project write-ups for the Experiment campaign were completed, as was an application for a grant from SYNENERGENE for advanced human practices.

Thursday, June 2

The Experiment online crowdfunding platform was submitted for approval in the morning, and an order for both competent cells and primers was placed. Background research was again the main focus, and most team members concentrated their efforts on learning the ins and outs of both Shewe (Shewanella oneidensis) and tiny moon phosphorus-eaters (Microlunatus phosphovorus).

Friday, June 3

This week’s conclusion was easily one of its most exciting moments, as the team took a field trip to visit Drs. Chi-hua Huang and Ashley Hammac of the USDA National Soil Erosion Research Lab, who generously donated both their time and expertise to answer questions and allow the team to tour the facility. Analytical labs, rain-producing machines for the purpose of studying erosion patterns, bioreactors, and anaerobic growth chambers were among the cutting-edge technologies the team observed. Thank you, Dr. Huang and Dr. Hammac!

Word was received later in the afternoon that the Experiment proposal had been approved; the campaign launches June 9!

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Monday, May 23

Day 1 on the job–the morning began with a tour of Bindley Bioscience Center for the new interns (Barrett, Caleigh, Emma, and Paige), during which Susan (the world’s best receptionist) kindly showed the fledgling researchers around and gave them the run-down on BBC do’s and don’t’s. The remainder of the day was spent working on gene design via Benchling, researching potential assays, and meeting with Drs. Rickus and Solomon. Important topics they raised included the importance of an experimental design plan, the need to begin with the basics (i.e. how do we know a protein is being expressed?) before delving into more complex issues of function, and gene design specifics.

Tuesday, May 24

Nine out of ten genes needed for luxury phosphorus uptake were ordered at the conclusion of Day 2! Throughout the day, genes were restructured for use with 3A Gibson Assembly protocol, and new constituent promoters were added (both sigma70 and sigmaS to arrest growth in P E. coli). Primer design began. Additionally, the hunt for assays continued for both phosphorus and nanowire constructs. Interns learned how to optimize codons using IDT both automatically and manually, checking for segments such as EcoRI, SpeI, XbaI, PstI, and HINDIII that should not be present in coding regions of constructs, as these sequences code for restriction sites.

Wednesday, May 25

A review of sterile technique and iGEM kit DNA extractions in the lab comprised a majority of the morning. Some golden rules of sterile technique: Always wipe down the lab bench with 70% ethanol before beginning; work as close to the flame as possible, as it creates a sterile “bubble”; and get into and out of pipette tip boxes as quickly as possible to keep them sterile. The autoclaving of new pipette tips and water was also used for demonstration purposes. Afternoon work consisted of continued definition of desired assays and design of nanowire genes.

Thursday, May 26

The creation of ready-to-use plates was the focus of lab work on Thursday. Interns followed an existing protocol to make plain LB agar, LB agar with ampicillin, and LB broth, culminating in the pouring and storing of too many plates to count. Caleigh, Sean, Suraj, and Paige took a field trip to the West Lafayette Waste Management facility to speak with Sara Peel of the Wabash River Enhancement Corporation (WREC) and Angela Andrews of the City of Lafayette Water Pollution Control. It was discovered that the facility is newly permitted for phosphorus removal; however, a chemical method as opposed to a biological one will be implemented August 1 for both political and financial reasons. The up-front cost for chemical treatment was less than for biological treatment, which made it the optimal choice for the time being, but the facility plans to switch to biological treatment as soon as it has the funds to do so, as this will be more cost-effective longterm. The team plans to return to the plant sometime in July to receive a full tour and collect wastewater samples to be used for testing.

wabash

 

Featured above is an overview of the Wabash River with the West Lafayette Wastewater Treatment Facility circled in red

Friday, May 27

Brainstorming for both background content for the official iGEM project wiki and a project timeline were completed in addition to an establishment of a protocol for completing lab notebooks. A Work Breakdown Structure (WBS) and Gantt Chart were constructed to record timeline information. Practice transformations of GFP and RFP kit plate parts were completed by the interns throughout the day. The conclusion of a successful first week!

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Welcome to the introductory post for the Purdue Biomakers’ 2016 iGEM project. Please look forward to weekly updates as we work toward the completion of our project in time for the Giant Jamboree in Boston, MA at the end of October.

Project Overview

Clean water is a fundamental necessity of human life. Thus, humans have developed methods for transforming dirty or polluted water into clean, safe water that can be used for a variety of purposes; however, traditional wastewater treatment does not recapture valuable components, such as bioavailable phosphorus. The 2016 iGEM project takes a holistic look at the water cycle and how synthetic biology can be implemented to improve water treatment. It was designed in two parts: A modular system for bioremediation, and expression of organic nanowires in E. coli for eventual use in energy generation and desalination. The initial inspiration and focus for the bioremediation system was the growing concern over bioavailable phosphorous. In combination with a separate interest in the creation of a more efficient microbial fuel cell (MFC), the fluffy white clouds of a dream took shape–a self-contained unit containing E. coli capable of processing wastewater by uptaking luxury amounts of phosphorus, nitrogen, and other impurities, generating electricity, and facilitating desalination. In goes grimy, opaque water, polluted by industry, agriculture, and daily living, and out comes clear, sparkling water, pure as alpine snow. Fantastic, right?

In taking steps to make this dream a reality, the team will be inserting DNA from phosphorus-accumulating organisms (PAOs) into E. coli to increase the phosphorus uptake of these microbial cells. Additionally, a separate E. coli strain will be engineered to include genes from Shewanella oneidensis (Shewe) in an effort to express organic nanowires, a feat that has never before been accomplished. Can we do it? Yes, we can! Below is a high-level timeline of major project milestones, and below that image is a photo of Shewe courtesy of Yuri Gorby, Rensselaer Polytechnic Institute, because it looks cool.

Proposed 2016 Project Timeline Shewanella with nanowires

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Monday, July 27th

On Monday morning we started off by meeting as a group to adapt our goals for the summer to the current progress of our experiments. We decided that we will aim for having all constructs ready to be transformed into yeast by the end of the summer period (Aug. 7th).

In addition, we began to insert the complete constructs that we had (AKR and MnP) into both the pSCB13C backbone (for submission to the iGEM Registry) and the pSB313 (for transformation into yeast) with restriction digests. Our yeast vector pSB313 contains an Amp resistance gene for easy cloning in E. coli, which would simplify later processing. We also replenished our gblock stocks by re-running PCR, since we used up most of the previous PCR amplification products with last week’s overlap PCR and Gibson experiments.

Tuesday, July 28th             

Tuesday we transformed both of the complete killswitch vectors and the AKR in pSB313 into E. coli. Since we wanted a simple method of characterizing/verifying our promoter for the killswitch, we plan to substitute the coding sequence in the killswitch with GFP and measure fluorescence; to do this, we also transformed a GFP sequence from the iGEM Registry for later 3A assembly. We also tried a new protocol for yeast transformation using all three of our backbones with fresh one-step buffer and parafilm, and we hope to see satisfactory growth by next week. As we continued to process our PCR reactions with gel extractions (as multiple bands often appeared on the image), we ran short of gel loading dye, but luckily we were able to borrow some from other Bindley occupants. Lastly, we had a meeting to discuss data representation and how we will design graphs to display our results.

Wednesday, July 29th

Wednesday we ran a Gibson reaction on Lignin Peroxidase and reorganized the freezer in the basement. For implementation into our yeast vector later, we digested the full killswitch device and pSB313. We also received our shipment of GelRed, a less toxic DNA stain for imaging our gels. Since the Lignin Peroxidase Gibson reaction only yielded one band on a gel, we decided to PCR purify the Gibson product. Finally, we obtained a wider gel comb which would allow us to load more DNA in a single gel run, increasing our DNA processing throughput, and we continued to gel extract gblock PCRs to replenish stocks.

Thursday, July 30th

On Thursday, we found out that there was growth on the negative controls for our killswitch/AKR transformation, and we believe it was because our LB + Chloramphenicol plates were improperly prepared. However, since we still had leftover transformants from the previous day, we poured new proper antibiotic plates and plated them. We extracted a PCR amplified Tyrosinase 1 and ran a Gibson assembly of the Tyrosinase, but a gel of the construct did not show any bands, we found out later in the day. We also decided to recheck a few of our DNA concentrations with Nanodrop since a smaller than expected concentration could result in the lack of bands; turns out the concentrations were in fact lower in the Gibson product of AKR, and an abundance of primers in the amplified Gibson distorted the reading. Thus, we ran another Gibson reaction for AKR.

Friday, July 31th

Friday we redid Gibson assemblies on Tyrosinase and Laccase and ran PCR amplification on the Lignin Peroxidase and Versatile Peroxidase full constructs. After checking them on gels, both Lignin Peroxidase and Versatile Peroxidase appear to be constructed correctly and ready for insertion into plasmid backbones. Tyrosinase and Laccase constructs should also be ready for insertion into vector backbones, which will be done over the weekend. By the middle of next week, we should be ready to submit all completed constructs in pSB1C3 to the iGEM Registry.

Posted

Monday, July 20th

We began the week by PCR amplifying three of the G blocks which had failed to adequately show up on a gel when we tried amplifying them the first time. Last Friday, we had begun trying to assemble G block 1 and 2 of the Manganese Peroxidase device by using overlap PCR. On Monday, we completed the overlap PCR of Manganese Peroxidase, but it was unsuccessful. After talking to our graduate adviser, Sam Lee, we found out that the annealing temperature we were using was probably too low. We changed the PCR program so that the annealing temperature was based off of the DNA overlap region’s melting temperature. Then, we began applying the new protocol to try and assemble VP1 and VP2 as well as the two AKR devices.

The killswitch team worked on ligating the promoter, RBS, lysing agent, and terminator.

The prototype team also met with Dr. Mosier to discuss the best way to design and build a model bioreactor. We now have a design and a list of parts to order.

Tuesday, July 21th

On Tuesday, we Nano-dropped the gel extraction we had done last week after overlap PCR amplification of Manganese Peroxidase and realized that it had been successful! We finally had an assembled device. Before we could transform the synthetic DNA into our yeast chassis, though, we needed to run a restriction enzyme digest and ligate the assembled MnP to our plasmid backbone. We completed that and then PCR amplified more G Blocks so that we could perform more Gibson Assembly later on in the week.

Also, the killswitch team transformed their entire assembled device into E. coli, so that the plasmids would replicate.

Wednesday, July 22th

The Manganese Peroxidase device which had been vector inserted on Tuesday was transformed into E. coli on Wednesday. Although overlap worked for Manganese Peroxidase, it did not work on any of the other G Blocks we tried. Thus, we decided to give overlap PCR a break and return to Gibson Assembly. We used our PCR purified AKR G Blocks from last week to run a Gibson Assembly. Also, since we were almost completely out of several PCR purified G Blocks, we ran a gel of our recent PCR amplified products and then PCR purified the ones which showed single bands at the desired base pair length.

Thursday, July 23th

On Thursday morning, the killswitch team purified the plasmids containing their killswitch device from the E. coli. They then Nano-dropped the results to make sure that their DNA concentration was high enough.

The enzyme track performed a Gibson Assembly of Versatile Peroxidase 1 and Versatile Peroxidase 2. We also gel extracted the PCR products of the G Blocks which had shown multiple bands in order to isolate the DNA at the desired base pair length. Finally, we ran a new gel to verify the remaining PCR products and to see if the Gibson Assembly of AKR had worked. The gel showed a faint band at the correct base pair length, so next we will work on ligating the device to our backbone.

Friday, July 24th

We were out of the lab for the entire day on Friday because we were visiting the Cardinal ethanol plant in Union City, Indiana. This visit was part of our Human Practices side of the project and helped give us a better idea of how current industrial ethanol plants work. The facility that we visited takes corn and produces 100 million gallons of ethanol each year. We were able to tour the facility and discuss our questions with the plant manager.

ethanol plant

 

 

 

 

 

 

 

 

The Purdue Biomakers touring the outside of the Cardinal Ethanol facility.

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Monday, July 13th

On Monday, the Enzyme Team was waiting on our dNTPs to arrive in order to PCR, but they never did. We used this time to further develop our project theme of pirates! Kill Switch team mini prepped and inoculated liquid cultures.

Tuesday, July 14th

On Tuesday, dNTPs had still not arrived, but the Enzyme Team was able to do a yeast transformation on Histine plates. We also continued our idea to make a prototype of a bioreactor by creating a prototype team and set up a meeting with Dr. Mosier. Kill switch team autoclaved and worked on developing our wiki.

Wednesday, July 15th

On Wednesday, our DNTPs finally arrived so we were able to PCR. Because our Gibson assembly did not work, we looked into PCR overlap, which would connect the two G blocks by using PCR. We PCR amplified, and ran a gel for manganese peroxidase, the first three wells being without primers, and the second three with. One of the bands with primers appeared, so the next day, we planned to run the rest of the manganese peroxidase. Kill switch team worked on mini-prep, restriction digest, and ligation.

Thursday, July 16th

On Thursday morning, we PCR amplified lignin peroxidase, gel extracted the manganese peroxide from the day before, and ran another gel of the rest of the manganese peroxide. The bands from the second gel were not in the correct place, so we did not gel extract. This may have been caused from problems with sterile technique or with the PCR overlap, so we decided to do another PCR overlap of lignin peroxidase. Kill switch team did transformations.

Friday, July 17th

On Friday morning, we ran a gel of lignin peroxidase in which nothing appeared. We looked into optimizing our PCR for PCR overlap and anything that might make something work. Enzyme team ended the day with more PCR and Kill Switch team was in “depression because nothing worked”.- Kate

Posted

This week’s update is a little bit different because the team has split into sub teams to be most effective in getting our project to completion. For now we have a group working on the killswitch part of our project and a team working with the G blocks of our enzymes. After the killswitch team completes their part, they will rejoin the rest of the group and work to take the project to completion

 

Monday, July 6th

Kill switch: completed a mini prep of the RBS, lysing agent, promoter and terminator with amazing results (above 300ng/ul) on the nanodrop. They also revamped the protocol for digestion and assembly to hopefully yield better results than the previous attempts

Enzyme: Spent the morning doing gel extractions and then testing their results on the nanodrop. Currently in a cycle of PCR, gel electrophoresis, gel extraction, then nanodrop. If the nanodrop results are less than 10 ng/ul repeat the cycle! All 14 G blocks need to make the cut so it is a lengthy process.

Tuesday, July 7th

Kill switch: a new ligation protocol was implemented today which had a lot more incubation time on the digestion (4 hrs) and ligation (16 hours). During our down time today we plated many things so that we had extra stocks of parts that we have already transformed this summer.

Enzyme: After repeating the cycle that we were in on Monday, we had some success! Our BLANK BLANK G Blocks had concentrations above our goal of 10ng/ul. And then we got back to the cycle so that all our G Blocks are above that 10ng/ul threshold.

Wednesday, July 8th

Kill Switch: Today we transformed the Promoter with RBS and the Lysing agent with the terminator into E. Coli making our first dual part transformation! Hopefully we’ve had enough practice and it was successful.  We also made a plethora of AMP plates because we were low. Lots of waiting around while things incubate and autoclave today.

Enzyme: Today we continued to PCR, run gels, image, extract, and test the concentrations of DNA with the nanodrop. We were able to get started with Gibson Assembly on the G Blocks that had concentrations of 10ng/ul or above from Tuesday!

Thursday, July 9th

Kill Switch: The dual part transformation from Wednesday wasn’t successful.  After a short mourning period we decided to re- ligate the digestion from Tuesday using some more tips from Sam and Janie.  Today was also filled with more house-keeping items, like autoclaving lots of equipment, and making broth for inoculations.

Enzyme: Electrophoresis gels were run on the Gibson assembly that was tried yesterday. Unfortunately, this Gibson was not successful. A practice yeast transformation was also performed.

Friday, July 10th

Kill Switch: Turns out that our transformations were successful! They just needed extra time to grow. Unfortunately, we already had ligated and used competent cells, so we decided to plate them anyway to see if the tips from Sam and Janie yielded better results.

Enzyme: After yesterdays failed Gibson assembly, we tried to do it again. Second times the charm, this time we used Sam as a resource and used ethanol precipitate to get rid of the elution buffer so that we could save the DNA.