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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.

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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

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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.

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Monday, June 29th

On Monday we continued PCRing the Gblocks and resuspended the new GBlock we received. We also got training to image the gels we ran on Friday. The kill switch team found that the second ligation transformation didn’t work and ran a gel of the digests to problem solve.

Tuesday, June 30th

On Tuesday we got gel results. Our PCR reactions did not have the expected results. Instead of having 1 band, the gels had smeared bands. Also, our digest gel only had one band per column, meaning the DNA was never cut. This would be why the ligation and transformation didn’t work.

Wednesday, July 1st

On Wednesday we learned that our PCR was not working because the thermo cycler was mislabeled. We were using the wrong sides of the machine. This was also the cause of the digest’s failure. We restarted PCR for all the Gblocks, doing it correctly this time. The kill switch team made more LB with Chloramphenicol to inoculate the promoter for miniprep. We received the terminator from the registry and transformed the lysing agent and terminator parts into E. Coli.

Thursday, June 2nd

On Thursday we continued doing correct PCR and running gels to confirm results. Additionally, we minipreped the promoter, RBS, and terminator parts for the kill switch. Also, we found that our terminator and lysing agent transformations had worked and inoculated some LB with Chloramphenicol so we would be set to miniprep and assemble on Monday.

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Monday, June 22nd

On Monday, we spent the day researching alternative assays to perform, as well as investigating alternative killswitch pathways. We also inoculated the E. Coli test colonies in LB+Chloromphenocol broth to determine the success rate of our promoter plasmids. Before plating the liquid culture, we also wrote out and clarified many of the procedures we’ve performed and others yet to come.

Tuesday, June 23rd

On Tuesday (upon the arrival of more buffers), we miniprepped the killswitch’s RBS, Backbone, and Terminator DNA sequences, preparing them for assembly. Also, our practice yeast transformation was discovered to be a failure, as our yeast broth was contaminated through unknown means. Bottlenecked by the lack of IDT DNA, we decided to re-write the majority of our procedures in paragraph form, for easier addition to our wiki.

Wednesday, June 24th

On Wednesday, we discovered yesterday’s miniprepping to be a failure, prompting a second effort, as well as miniprepping the promoters for eventual transformation. Also completed today was the first preparation of our switchgrass sample via milling. We began the transformation of our RBS+Terminator genes into competent E. Coli cells. Outside of the lab, we began brainstorming fitting titles for our project. Considering the Pirate theme, “Dead Lignin Tell No Tales” seems to be the current leader.

Thursday, June 25th

On Thursday, our G-Blocks arrived from IDT! We spent the afternoon diluting and re-suspending the DNA samples, and began PCR in the late afternoon. We also started our second attempt at the RBS+Terminator assembly (as Wednesday’s failed… the use of competent cells places our DNA under suspicion.). We also began electrophoretic gel construction, in order to more fully check our results and discover the source of the error.

Friday, June 26th

On Friday, we underwent gel imaging training, in order to obtain clearer results from the gels (the ethidium bromide used in the gel fluoresces under UV light, allowing easy identification of DNA bands). Then, we ran gels for last night’s PCR products. We also poured more LB/LB+Chloromphenocol broth, for use when testing the transformations of E. Coli ligations (also performed today). We also discovered that the assembly of the RBS to the terminator was a failure, prompting the research and purchasing of new killswitch components. Also, due to our Graduate advisor running a workshop next week (rendering BIND134 unusable), we moved supplies downstairs for easy access.

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Monday, June 15th

On Monday we started our day greeting our buddy high schoolers that came from the MASI program at Purdue. We showed them through basic lab sterile technique and got their aid on our second, revised mini prep, which gave a high yield of our E.coli. Backbones. In addition, we finally managed to tie up loose ends with our gene problems and ordered all the parts we needed from the registry and IDTDNA.

Tuesday, June 16th

On Tuesday we discussed our project progress with our PI, Jenna Rickus, and got her input on how to assay our enzymes that were transformed in our yeast. We then began running a growth curve test on our wild type yeast cells to set a standard to compare our modified yeast to. However, complications began to rise when the machine we used to count or cells experienced issues. Due to technical difficulties, we had to postpone this test for a later time. The high schoolers got the chance to learn more about lab technique when we decided to start our first E.coli transformations with our promoters, RBS, amp backbone, lysis agent, and terminator genes.

Wednesday, June 17th

On Wednesday we went through a second round of E.coli. Backbone mini prep in order to stockpile on DNA for future experiments. However, due to restraints on mini prep supplies at the end of our second round, we resorted to creating our own buffers in the lab. In addition, only one of the transformations that we did yesterday went through successfully. Because of this, we were really bottlenecked for tasks and experiments we could do. Instead, we turned to coming up with possible themes for our lignin breakdown project, writing up more of our wiki information, and started on our yeast death curve.

Thursday, June 18th

On Thursday, we decided to attempt another E.coli backbone mini prep with our new buffers, but faced some lackluster results as the buffer we made was not optimal enough to extract the DNA properly. Also, the re-cultured E.coli. Were submersed in broth that didn’t have antibiotic resistance, so selective pressures reduced the amount of DNA that we could collect from a sample. Since we had to wait for our transformed cells to grow in their plates, the only lab related activity we had left was to create more competent cells for future experiments. The MTT assay kit we had bought also had very vague and confusing instructions, so we needed to refer to our graduate assistants and advisors for help and alternatives for the assay.

Friday, June 19th

The majority of the work day involved running additional transformations with our yeast parts and mini prepping the promoter plasmids from E.coli liquid cultures. However, due to selective pressures and inefficient buffers, the mini prepping needed to be put on hold until more company buffers could be ordered. The rest of the time was spent working on the wiki, organizing our timeline, and researching future assays.

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Monday, June 8th

On Monday we helped Soo pour plates to prepare for her projects and worked on protocols. Additionally, we began to organize our pages for our wiki and started protein profiles. We also discussed our killswitch ideas and worked to find the gene sequence for our second killswitch promoter.

Tuesday, June 9th

On Tuesday we worked on G-Blocks and promoters and met with Sam to figure out how we would make our primers. We worked on our G-Blocks, started on primers, and continued working on our materials list. We also continued work on our protein profiles.

Wednesday, June 10th

On Wednesday we met with Sam and Janie to discuss our “finalized” G-Blocks. We learned that our proposed method of gblock construction was not the best as having the prefix and suffix before each part would stop us from being able to submit our minal design as a full part.,  They both discussed our tagging methods for cellulase and xylanase and at the meeting we decided to use antibody tagging for these proteins. We also learned that we would need to redesign our gblocks as yeast is unable to produce multiple proteins with only one RBS. We redesigned our system to have a Kozak sequence before both the lignin breakdown enzyme and the helper enzyme. Finally, we decided to cut cellulase and xylanase from our design because of cost and just order the enzymes for testing.

Thursday, June 11th

On Thursday we helped Soo with her GERI students and epidemics presentation. It was a fun and rewarding experience and was good outreach for the iGEM team. Unfortunately we were forced to cancel our G-Block order as we had chosen a green florescent protein that was incomplete. We researched nduring the day to find a new GFP and possibly a new secretion tag in case we found a workable GFP designed for a different terminus. We ended up finding a possible replacement but the documentation was unclear and we decided to hold off on ordering until we talked to Dr. Rickus.

Friday, June 12th

Friday was factually the best day of the week. In the first hour we redesigned our primers and had them ready to go. Early in the day Dr. Rickus stopped by to talk to us about our progress and we were able to discuss our GFP progress. She was able to help us select our GFP and talked to us about linkers which we then researched and added to our parts list. We learned that our primers were no longer god as we needed to use a different iGEM prefix to meet reguloations and so we changed our G-Block design. Shortly thereafter we began to construct our final G-Blocks and then moved on to our primers. We started miniprep but some issues arose and we moved the miniprep to Monday. Before concluding for the day we ran our primers and Gblocks through a series of checks to ensure we would be able to complete PCR and that our sequences were correct. We ordered our DNA and ended the day on a good note.