Team:Calgary/Notebook
NOTEBOOK
CELLULASE: May
Cellulase | Omega/thymol | Biosafety | Hardware | Software
May 4 - 8
This was our first full week of iGEM Calgary 2020! We narrowed down the final project ideas to Biomining, Cow Herpes, Mycotoxins, and Mycorrhizae. Through information gathered from our literature reviews and Human Practices meetings, we also began to look into Johne’s Disease as a potential project area, but ultimately eliminated both Cow Herpes and Johne’s Disease. By the end of the week, we turned our attention only to Mycorrhizae, Mycotoxins, and Biomining.
With regards to mycotoxins, we began literature review to determine the need for and feasibility of a lactobacillus probiotic that contains degradation enzymes for major mycotoxins found in animal feed. In biomining, we originally had the idea to use a purified lanthanide binding protein, Lanmodulin, to bind the metals. However, following further literature review and Human Practices meetings, we decided that it is more feasible to express the protein on the cell surface of a genetically engineered microorganism (lower cost) or engineer our own binding tag/binding protein hybrid and display that on the cell surface.
Throughout the week, our dry lab members were hard at work brushing up on their software skills and brainstorming innovative hardware and software ideas for each potential project area. In addition, our team began working on graphic design for our educational tools! Finally, we ended our week with a virtual team building workshop, where we completed small group challenges and developed the 6 Rules of iGEM Calgary.
May 11 - 15
With our focus mainly on Biomining, Mycotoxins, and Mycorrhizae this week we went into full swing with HP meetings to narrow in on a project idea. We also had our first Synbiolympics event!
For Mycotoxins we had a meeting with a couple researchers from the University of Saskatchewan and a dairy cow veterinarian. We learned from our HP meetings that this problem may not be very relevant in Alberta and that there seem to already be effective solutions for it. Specifically, a company called Biomin has a lot of products and patents surrounding mycotoxins handling. We determined we need to do more searching to see if this project was worth it. Despite promising papers and HP meetings with a few professors, the Mycorrhizae project was unfortunately abandoned as the scope was determined to be too big and out of our capabilities. From the HP meetings last week Biomining had been having some patent troubles so we were trying to see if we could change the proteins enough to get around the patents. Bioleaching also became a new avenue to explore.
While also participating in literature review and HP, our dry lab members continued to learn new software programs and skills and they started a project called Bellatrix. A new idea was also proposed to look at DIY lab equipment.
With our main three project ideas from last week dwindling in support we decided to have a larger brainstorm session to look into new ideas. We had pitches relating to bacterial cellulose for skin care, miRNA or peptides for anti-inflammatory protection, probiotic gum to treat dental caries, silica nanoparticle-producing algae, and yeast supplements for vitamin A deficiency.
May 19 - 22
This week our team branched off into many little subgroups to focus on the various new ideas we pitched last week. In addition, we had our first education meeting to discuss our many ideas for education, and also started to get some other subgroups forming like JulyGEM and cGEM.
All of the subgroups had worked on fleshing out their ideas and doing some preliminary HP and patent analysis. However, we started making some larger team decisions on whether we should really allow patents to limit our ideas as we had a meeting with a lawyer who suggested otherwise. The team is definitely feeling anxious to pick a project and with funding deadlines approaching we decided to set a deadline for next week.
In addition to literature review and HP, we continued our other work in graphic design for the education slides and software skill development.
May 25 - 29
We had a lot of long meetings this week but we finally picked a project!! After a few more HP meetings and internal meetings discussing feasibility of certain ideas we narrowed down to four to be presented as contenders for 2020 project.
After the continued research of this week and the various HP meetings we narrowed in on four ideas: Bacterial Cellulose Skin Care, Biomining/Bioleaching, Probiotic Gum for Dental Caries, and a sustainable GMO Yeast Supplement for Vitamin A deficiency. Each group presented in lengths the pros and cons of the projects, outlining the prospects for HP and dry lab, while also critically analyzing the feasibility for wet lab and the marketability. After this very long discussion we conducted a vote and decided that the GMO Yeast Supplement for Vitamin A Deficiency would be our project.
At the end of the week we started looking more into the wet lab components and how to do the genetic manipulation itself as this is a new chassis for us. Dry lab also brainstormed projects they could start working on for this and we had preliminary discussions regarding modelling possibilities. Our next goal is to get more HP meetings going, do some more research for the wet lab, and start focusing on other subgroups like JulyGEM, Funding, cGEM, Education, and the Faculty Talk.
SOFTWARE: May
Cellulase | Omega/thymol | Biosafety | Hardware | Software
May 4 - 8
This was our first full week of iGEM Calgary 2020! We narrowed down the final project ideas to Biomining, Cow Herpes, Mycotoxins, and Mycorrhizae. Through information gathered from our literature reviews and Human Practices meetings, we also began to look into Johne’s Disease as a potential project area, but ultimately eliminated both Cow Herpes and Johne’s Disease. By the end of the week, we turned our attention only to Mycorrhizae, Mycotoxins, and Biomining.
With regards to mycotoxins, we began literature review to determine the need for and feasibility of a lactobacillus probiotic that contains degradation enzymes for major mycotoxins found in animal feed. In biomining, we originally had the idea to use a purified lanthanide binding protein, Lanmodulin, to bind the metals. However, following further literature review and Human Practices meetings, we decided that it is more feasible to express the protein on the cell surface of a genetically engineered microorganism (lower cost) or engineer our own binding tag/binding protein hybrid and display that on the cell surface.
Throughout the week, our dry lab members were hard at work brushing up on their software skills and brainstorming innovative hardware and software ideas for each potential project area. In addition, our team began working on graphic design for our educational tools! Finally, we ended our week with a virtual team building workshop, where we completed small group challenges and developed the 6 Rules of iGEM Calgary.
GausHaus development began and the script for cleaning up dynamic pdbs was begun.
May 11 - 15
With our focus mainly on Biomining, Mycotoxins, and Mycorrhizae this week we went into full swing with HP meetings to narrow in on a project idea. We also had our first Synbiolympics event!
For Mycotoxins we had a meeting with a couple researchers from the University of Saskatchewan and a dairy cow veterinarian. We learned from our HP meetings that this problem may not be very relevant in Alberta and that there seem to already be effective solutions for it. Specifically, a company called Biomin has a lot of products and patents surrounding mycotoxins handling. We determined we need to do more searching to see if this project was worth it. Despite promising papers and HP meetings with a few professors, the Mycorrhizae project was unfortunately abandoned as the scope was determined to be too big and out of our capabilities. From the HP meetings last week Biomining had been having some patent troubles so we were trying to see if we could change the proteins enough to get around the patents. Bioleaching also became a new avenue to explore.
While also participating in literature review and HP, our dry lab members continued to learn new software programs and skills and they started a project called Bellatrix. A new idea was also proposed to look at DIY lab equipment.
With our main three project ideas from last week dwindling in support we decided to have a larger brainstorm session to look into new ideas. We had pitches relating to bacterial cellulose for skin care, miRNA or peptides for anti-inflammatory protection, probiotic gum to treat dental caries, silica nanoparticle-producing algae, and yeast supplements for vitamin A deficiency.
GausHaus has yet to be trained but now the pdb coordinates are readily available and usable in the algorithm.
May 19 - 22
This week our team branched off into many little subgroups to focus on the various new ideas we pitched last week. In addition, we had our first education meeting to discuss our many ideas for education, and also started to get some other subgroups forming like JulyGEM and cGEM.
All of the subgroups had worked on fleshing out their ideas and doing some preliminary HP and patent analysis. However, we started making some larger team decisions on whether we should really allow patents to limit our ideas as we had a meeting with a lawyer who suggested otherwise. The team is definitely feeling anxious to pick a project and with funding deadlines approaching we decided to set a deadline for next week.
In addition to literature review and HP, we continued our other work in graphic design for the education slides and software skill development.
GausHaus has been trained! Now we are looking to develop what matrix and vector to use for the biggest impact.
May 25 - 29
We had a lot of long meetings this week but we finally picked a project!! After a few more HP meetings and internal meetings discussing feasibility of certain ideas we narrowed down to four to be presented as contenders for 2020 project.
After the continued research of this week and the various HP meetings we narrowed in on four ideas: Bacterial Cellulose Skin Care, Biomining/Bioleaching, Probiotic Gum for Dental Caries, and a sustainable GMO Yeast Supplement for Vitamin A deficiency. Each group presented in lengths the pros and cons of the projects, outlining the prospects for HP and dry lab, while also critically analyzing the feasibility for wet lab and the marketability. After this very long discussion we conducted a vote and decided that the GMO Yeast Supplement for Vitamin A Deficiency would be our project.
At the end of the week we started looking more into the wet lab components and how to do the genetic manipulation itself as this is a new chassis for us. Dry lab also brainstormed projects they could start working on for this and we had preliminary discussions regarding modelling possibilities. Our next goal is to get more HP meetings going, do some more research for the wet lab, and start focusing on other subgroups like JulyGEM, Funding, cGEM, Education, and the Faculty Talk.
Gaushaus has been trained on complete data and has been used to produce new coordinates.
SOFTWARE: June
Cellulase | Omega/thymol | Biosafety | Hardware | Software
June 1 - 4
The Vitamin-A Deficiency map project was started. This included coding Python scripts to visualize and predict VAD levels across the world. Logistic regression algorithm was planned to be used for this program. We realized it was important to understand some of the metabolic pathways of our project .We conducted literature review on flux balance analysis, and then metabolic flux analysis. PDB file handling was automated for Bellatrix, so we could download directly from Protein Data Bank. We developed a new visualization method ‘sticks to see the protein work within R. Cellulase sequence research began today.
June 8 - 12
More work on the Vitamin-A Deficiency map was done, which led to the conclusion that this subproject would be discontinued due to a significant lack of available data on VAD.
Bellatrix research was done regarding how we could leverage Pandas data structures in our code. We ran into issues dealing with PDB files, as they apparently seem to be unorganized and nonstandard. GausHaus was put on hold to focus on the cellulase modelling and engineering.
June 15 - 19
Research was done on Metabolic Flux Analysis/Flux Balance Analysis in relation to optimizing yield. Other mathematical models were also considered. We studied the format of a PDB file, identified common factors in a PDB file that make it hard to understand the data, and then wrote functions to allow the user to both understand and utilize PDB data. This was implemented into Bellatrix.
June 22 - 26
Wiki subgroup started to get organized. Research and practice was done on HTML and CSS to prepare for coding in the coming weeks. We expanded Bellatrix to run on an interrogation set of proteins. A Star file was brainstormed and we attempted to translate this into code.
June 29 - July 3
The Wiki subgroup created various imposter pages to begin to develop a framework for our Wiki. This included a imposter landing page, content page, team page and journal page.
SOFTWARE: July
Cellulase | Omega/thymol | Biosafety | Hardware | Software
July 6 - 10
The Wiki subgroup devised a plan to restructure the Wiki code to be more coder-friendly. This included the separation of HTML and CSS, as well as the creation of various template stylesheets and pages.
Work on the team page began this week. Bellatrix also neared completion. We structured the Stars files, and implemented exception handling to prevent program failure. Optimizations were added to decrease the amount of time it took to construct Bellatrix libraries. SEGI-8 dynamics were started and completed
July 13 - 17
The Wiki subgroup created organizational documents to help support the team in Wiki development. This included general styling instructions as well as instructions to use/edit the navbar.
Metabolic flux analysis was determined to be performed without experimental data. The way a Star file represents its coordinates was changed based on feedback from a protein modeller. EGI dynamics were started and completed
July 20 - 24
A new project called PEGI (Predicted Essential Gene Identification) started, and initial research begun.
The Wiki subgroup spent the week debugging and improving our imposter pages. Gene knockouts were determined to be useful in PEGI. We worked on developing schema for integrating Bellatrix into modelling.
July 27 - 31
The PEGI subgroup devised an action plan for more in-depth research into how we could leverage neural network technology to predict essential genes.
The Wiki subgroup got feedback from branding and implemented various styling changes to the Wiki framework. We investigated using Stars as a way to improve BLAST’s usefulness, by clustering proteins returned from the list found by a BLAST search. We looked into clustering methods.
SOFTWARE: August
Cellulase | Omega/thymol | Biosafety | Hardware | Software
August 3 - 7
The Wiki subgroup finished off another draft of the landing page, as well as worked on improving the notebook menu and sidebar. Cobrapy work was done to identify essential genes in R. toruloides Dimensionality reduction techniques were researched to try and implement in clustering using Bellatrix’s Star files. SEGI-8 dynamics were redone with new protein structure.
August 10 - 14
The Wiki subgroup continued to make changes based on feedback from branding. The visual aesthetic of the Wiki was being pulled together by this point. The team page was reworked in accordance to the branding team’s suggestions.
August 17 - 21
The Attributions page was made.
August 24 - 28
Minor revisions to the team page and Attributions page were made. The bioreactor team looked into the mathematical modelling involved in the reactor design and methods of harvesting. Bellatrix was modified to allow users to implement custom PDB files. Chi angles were attempted to be added to a Star file.
August 30 - September 3
The PEGI subgroup disbanded to focus on Wiki work.
SOFTWARE: September
Cellulase | Omega/thymol | Biosafety | Hardware | Software
September 6 - 10
The Wiki subgroup spent this week fixing various bugs on the developed pages. For example, we added mobile configuration so that the sidebar will hide away on small screens, allowing more room for content. Also, the notebook page was updated to be responsive. This means that the buttons are now functional and show the correct content upon click.
The Bellatrix Manuscript group also made some progress. Our paper was submitted for feedback to The Journal Initiative, where in exchange for corrections on our work, we provided feedback for two other papers. We reviewed a paper on Rosetta as well as a paper on a project called “Mossphate”. Valuable feedback was obtained for the Bellatrix manuscript through this process.
Peer review on University of Helsinki’s “Guide on how to use Rosetta”, and University of Dusseldorf’s “Mossphate” were done. Edits/suggestions were made to each paper.
Modelling this week worked on generating the lipid bilayer models necessary for Omega threes. Alpha linoleic acid was used with a small relaxed bilayer structure. With KCL Multiple Homology models were generated using Chimera for the team. Along with this writing began for aspects of GAUSHAUS.
September 13 - 17
Some revisions to the team page code were made to fix issues. The Attributions banner page has been fixed and is now ready for input. The individual photos were updated as well using the photos taken from last weekend.
Worked on Bellatrix briefly, trying to get it to read different file types. We were able to verify that the parsing of a PDB file into a pandas dataframe works for dynamic data. We started to work towards integrating this option into the Bellatrix GUI to enable more functionality. We found that the issue with loading in dynamic data is the size. Star files take a PDB file and make a more descriptive, yet exponentially bigger file. The issue is that the Python program that builds this file does not have enough access to RAM to create this file in local memory before writing it to a csv file. We decided to only use one row of these Star files. For context, this dynamic data file holds 44,000 residue coordinates. To make an nxn matrix, that would require 1936000000 doubles (variables). This is simply too large to make a practical file.
Manuscript began to collect the information provided by our reviewers Modelling began working on models for Michas antiworm proteins, the first is fondly nicknamed Terpy1. At this stage just some sequence stuff with a prelim BLAST and multi sequence alignment. Along with this modelling worked on a poster for the PURE committee. For this poster the first AllForOne protein visualization was created.
September 20 - 24
The Wiki subgroup continued to work on bug fixes and updates. The Navigation bar and Footer were updated with 2020 links and are now ready to use. On top of this, a variety of sizing issues were remedied. More work was done on content writing as well.
There was an issue with the modal layout when the page is resized on computer browsers. This has been fixed as of this week. The subprojects section in the modal still needs some work and we are currently waiting for the graphics.
This week iGAM was modified for use on the KCL protein, a temporary fitness function was utilized while the team looked to determine a piecewise function that would work well with the desired half lives. Also assisted the team with relaxation and prep of their structure. Terpy1 Modelling continued looking to develop MD for the protein. First though New blasts and Homology structures are needed that are more trustworthy.
September 27 - October 1
Modelling worked on rerunning dynamics for the cellulases dynamics for standardized measurement so that we can utilize some controls. Along with this there was a meeting to determine the graphic design necessary Sticks™ code was isolated and manual was begun. Manuscript subgroup began fulfilling reviewer wishes for final submission.
SOFTWARE: October
Cellulase | Omega/thymol | Biosafety | Hardware | Software
October 4 -8
The Wiki subgroup made changes to the landing page, including the addition of project navigation tiles which betters the flow of the Wiki. Additionally, some journal entries were added in.
October 11 - 15
The Wiki subgroup is in high gear finishing up pages and writing content.
October 18 - 19
Wiki writing.
HARDWARE: May
Cellulase | Omega/thymol | Biosafety | Hardware | Software
May 4 - 8
This was our first full week of iGEM Calgary 2020! We narrowed down the final project ideas to Biomining, Cow Herpes, Mycotoxins, and Mycorrhizae. Through information gathered from our literature reviews and Human Practices meetings, we also began to look into Johne’s Disease as a potential project area, but ultimately eliminated both Cow Herpes and Johne’s Disease. By the end of the week, we turned our attention only to Mycorrhizae, Mycotoxins, and Biomining.
With regards to mycotoxins, we began literature review to determine the need for and feasibility of a lactobacillus probiotic that contains degradation enzymes for major mycotoxins found in animal feed. In biomining, we originally had the idea to use a purified lanthanide binding protein, Lanmodulin, to bind the metals. However, following further literature review and Human Practices meetings, we decided that it is more feasible to express the protein on the cell surface of a genetically engineered microorganism (lower cost) or engineer our own binding tag/binding protein hybrid and display that on the cell surface.
Throughout the week, our dry lab members were hard at work brushing up on their software skills and brainstorming innovative hardware and software ideas for each potential project area. In addition, our team began working on graphic design for our educational tools! Finally, we ended our week with a virtual team building workshop, where we completed small group challenges and developed the 6 Rules of iGEM Calgary.
May 11 - 15
With our focus mainly on Biomining, Mycotoxins, and Mycorrhizae this week we went into full swing with HP meetings to narrow in on a project idea. We also had our first Synbiolympics event!
For Mycotoxins we had a meeting with a couple researchers from the University of Saskatchewan and a dairy cow veterinarian. We learned from our HP meetings that this problem may not be very relevant in Alberta and that there seem to already be effective solutions for it. Specifically, a company called Biomin has a lot of products and patents surrounding mycotoxins handling. We determined we need to do more searching to see if this project was worth it. Despite promising papers and HP meetings with a few professors, the Mycorrhizae project was unfortunately abandoned as the scope was determined to be too big and out of our capabilities. From the HP meetings last week Biomining had been having some patent troubles so we were trying to see if we could change the proteins enough to get around the patents. Bioleaching also became a new avenue to explore.
While also participating in literature review and HP, our dry lab members continued to learn new software programs and skills and they started a project called Bellatrix. A new idea was also proposed to look at DIY lab equipment.
With our main three project ideas from last week dwindling in support we decided to have a larger brainstorm session to look into new ideas. We had pitches relating to bacterial cellulose for skin care, miRNA or peptides for anti-inflammatory protection, probiotic gum to treat dental caries, silica nanoparticle-producing algae, and yeast supplements for vitamin A deficiency.
May 19 - 22
This week our team branched off into many little subgroups to focus on the various new ideas we pitched last week. In addition, we had our first education meeting to discuss our many ideas for education, and also started to get some other subgroups forming like JulyGEM and cGEM.
All of the subgroups had worked on fleshing out their ideas and doing some preliminary HP and patent analysis. However, we started making some larger team decisions on whether we should really allow patents to limit our ideas as we had a meeting with a lawyer who suggested otherwise. The team is definitely feeling anxious to pick a project and with funding deadlines approaching we decided to set a deadline for next week.
In addition to literature review and HP, we continued our other work in graphic design for the education slides and software skill development.
May 25 - 29
We had a lot of long meetings this week but we finally picked a project!! After a few more HP meetings and internal meetings discussing feasibility of certain ideas we narrowed down to four to be presented as contenders for 2020 project.
After the continued research of this week and the various HP meetings we narrowed in on four ideas: Bacterial Cellulose Skin Care, Biomining/Bioleaching, Probiotic Gum for Dental Caries, and a sustainable GMO Yeast Supplement for Vitamin A deficiency. Each group presented in lengths the pros and cons of the projects, outlining the prospects for HP and dry lab, while also critically analyzing the feasibility for wet lab and the marketability. After this very long discussion we conducted a vote and decided that the GMO Yeast Supplement for Vitamin A Deficiency would be our project.
At the end of the week we started looking more into the wet lab components and how to do the genetic manipulation itself as this is a new chassis for us. Dry lab also brainstormed projects they could start working on for this and we had preliminary discussions regarding modelling possibilities. Our next goal is to get more HP meetings going, do some more research for the wet lab, and start focusing on other subgroups like JulyGEM, Funding, cGEM, Education, and the Faculty Talk.
HARDWARE: June
Cellulase | Omega/thymol | Biosafety | Hardware | Software
June 1 - 4
Possible low-cost solutions for the field bioreactor designs were researched. The team looked into designs that can be easily implemented anywhere.
June 8 - 12
The team decided that an airlift bioreactor would be the best design to follow as it is simpler than the other designs.
June 15 - 19
The team looked into inexpensive and simple air pump designs to be attached to the bioreactor.
June 22 - 26
Ideation of the bioreactor designed for laboratory use started. It was decided that this design would also be an airlift bioreactor.
HARDWARE: July
Cellulase | Omega/thymol | Biosafety | Hardware | Software
July 6 - 10
We began looking into vitamin A indicators in the blood through literature. There seems to be high potential for identifying VITA deficiency through retinol binding protein. In addition, there began study of electrochemical cells, which the Randle Circuit models, to create a model that we could use for our device. We encountered difficulties when it was realized the many simplifications and alterations we can make depending on the operating conditions of the circuit. We are still identifying these operating conditions and will continue to look into electrochemical cell monitoring. Ohmmeter were also investigated, as this will be an integral part of our project design. To make it cheap, we need to create our own. This may prove difficult as these are precision instruments.
Discussions of ideas started regarding possible bioreactor designs.
July 13 - 17
After running into a roadblock, we managed to verify the importance and viability of this project. We are scheduling appointments to meet with professors to develop a proprietary measurement device for our VITA tests. Most of our time was spent on literature review to identify existing methods and select the best way for detecting RBP.
July 20 - 24
We continued research into the biochemistry behind the randle cell. This led to discoveries of important considerations to make. Things such as electrode size, cell tolerance of voltage and diffusion all needed to be taken into account. We will find the limiting constraints the next week. We also looked for HP contacts to use for reference and verify our design.
July 27 - 31
This week saw many design changes, and a lot of project consideration. Notably, we met with Dr. Colin Dalton and his PhD student, who gave invaluable insight on our device design, as well as the socioeconomic factors that we must consider. They ended up providing us with verification that our design would work, and recommended that we use plasmids instead of antibodies, due to their lower cost and better shelf-life. They pointed out that this project would look something like a year long, and that it requires a lot of precise instruments. Precision and confidence in the technology we were using could make or break this project. We worked on some online videos elaborating on how our intended IC, the AD 9533, works so that I may use this understanding when designing the circuit.
HARDWARE: August
Cellulase | Omega/thymol | Biosafety | Hardware | Software
August 10 - 14
Made a lot of progress this past week. I poured over the documentation for the AD5933 chip. We did research into the theory behind its operation. This included digital to analog converters and waveform generation using a direct digital synthesizer. Mac and Randy worked on setting up the circuit together. They got so far as to begin to code the chip to allow us to use it! We are currently working through the code, troubleshooting and assessing its function, as we were able to find open-source code already made by an individual.
August 17 - 21
No notable work was done on the Randle Cell
August 24 - 28
We spent time looking through literature on impedance based diagnostic tests as well as understanding the code for the IC.
August 30 - September 3
No notable work was done.
HARDWARE: September
Cellulase | Omega/thymol | Biosafety | Hardware | Software
September 6 - 10
We reached out to multiple members of the BioMEMS and Bioinspired Microfluidic Laboratory to try and get feedback on our design. We received no responses and will continue to contact researchers.
September 13 - 17
Trying to make our approach more realistic, as well as useful, I reached out to various experts in microfluidics and lab-on-a-chip diagnostics to get feedback and assess the probability of using blood droplets, rather than whole blood. Literature suggested that it was possible to detect RBP in plasma using dried blood samples. This, however, requires an intensive laboratory procedure to use dried blood.
I investigated our current strategy of biotin-streptavidin binding as the mechanism to immobilize the aptamer on the electrode. Silane reagents appear to be inadequate due to the lack of selectivity, which could interfere with impedance results.
September 20 - 24
I searched up things we need to consider for the bioreactor (i.e. values we need to determine/test using the bioreactor). After looking it up and asking another team member from wet lab, I determined that spectroscopy could be something we can use to determine the experimental growth rate in the bioreactor. I also sent out an email to PBS Biotech, but they said they wouldn’t be able to help us as they do not specialize in yeast. They suggested that we contact breweries as they may be able to help us with yeast-specific bioreactor questions.
We met to interpret and verify the impedance measurements we were reading from our device. We were able to illicit changes in the impedance by introducing different resistors to the output. We were unable to get correctly calibrated measurements however. We believe that the error lies in the calibration of the IC (Integrated Circuit) that we purchased. To effectively measure a target impedance, we must change the value of a resistor on the board to switch the chip into a different mode. This led to important research into understanding how the signal is amplified, converted between analog and digital, and finally measured using the Discrete Fourier Transform. We also found multiple softwares existing that we can use to expedite the coding progress. We still have to make sense of some of this code. Mac and I also ended up working on the bioreactor design, and we found some fluid dynamics software that we are going to use. We also ended up switching to Arduino Uno as the microcontroller instead of the Teensy for technical issues
September 27 - October 1
Met with a PhD student (Sultan Khetani) to discuss biosensors, and mechanisms to identify target biomarkers. A very useful resource. He basically verified that our design can work. He mentioned that aptamers are typically found by a specific process of elimination, and if our aptamer sequence was found using Selex, then it should be able to select RBP from whole blood. This is great news as this eliminates a step in our diagnostic process. He recommended that we look into other ways of measurement besides blood (urine, saliva, etc). He also gave us a list of people that he recommends as mentors for this project, and offered to let us shadow his lab. We intend to shadow some members working in the BioMEMs laboratory at U of C.
HARDWARE: October
Cellulase | Omega/thymol | Biosafety | Hardware | Software
October 5 - 9
October content
October 12 - 16
October content
October 19 - 23
October content
October 26 - 27
WIKI WRITING
OMEGA: May
Cellulase | Omega/thymol | Biosafety | Hardware | Software
May 4 - 8
This was our first full week of iGEM Calgary 2020! We narrowed down the final project ideas to Biomining, Cow Herpes, Mycotoxins, and Mycorrhizae. Through information gathered from our literature reviews and Human Practices meetings, we also began to look into Johne’s Disease as a potential project area, but ultimately eliminated both Cow Herpes and Johne’s Disease. By the end of the week, we turned our attention only to Mycorrhizae, Mycotoxins, and Biomining.
With regards to mycotoxins, we began literature review to determine the need for and feasibility of a lactobacillus probiotic that contains degradation enzymes for major mycotoxins found in animal feed. In biomining, we originally had the idea to use a purified lanthanide binding protein, Lanmodulin, to bind the metals. However, following further literature review and Human Practices meetings, we decided that it is more feasible to express the protein on the cell surface of a genetically engineered microorganism (lower cost) or engineer our own binding tag/binding protein hybrid and display that on the cell surface.
Throughout the week, our dry lab members were hard at work brushing up on their software skills and brainstorming innovative hardware and software ideas for each potential project area. In addition, our team began working on graphic design for our educational tools! Finally, we ended our week with a virtual team building workshop, where we completed small group challenges and developed the 6 Rules of iGEM Calgary.
May 11 - 15
With our focus mainly on Biomining, Mycotoxins, and Mycorrhizae this week we went into full swing with HP meetings to narrow in on a project idea. We also had our first Synbiolympics event!
For Mycotoxins we had a meeting with a couple researchers from the University of Saskatchewan and a dairy cow veterinarian. We learned from our HP meetings that this problem may not be very relevant in Alberta and that there seem to already be effective solutions for it. Specifically, a company called Biomin has a lot of products and patents surrounding mycotoxins handling. We determined we need to do more searching to see if this project was worth it. Despite promising papers and HP meetings with a few professors, the Mycorrhizae project was unfortunately abandoned as the scope was determined to be too big and out of our capabilities. From the HP meetings last week Biomining had been having some patent troubles so we were trying to see if we could change the proteins enough to get around the patents. Bioleaching also became a new avenue to explore.
While also participating in literature review and HP, our dry lab members continued to learn new software programs and skills and they started a project called Bellatrix. A new idea was also proposed to look at DIY lab equipment.
With our main three project ideas from last week dwindling in support we decided to have a larger brainstorm session to look into new ideas. We had pitches relating to bacterial cellulose for skin care, miRNA or peptides for anti-inflammatory protection, probiotic gum to treat dental caries, silica nanoparticle-producing algae, and yeast supplements for vitamin A deficiency.
May 19 - 22
This week our team branched off into many little subgroups to focus on the various new ideas we pitched last week. In addition, we had our first education meeting to discuss our many ideas for education, and also started to get some other subgroups forming like JulyGEM and cGEM.
All of the subgroups had worked on fleshing out their ideas and doing some preliminary HP and patent analysis. However, we started making some larger team decisions on whether we should really allow patents to limit our ideas as we had a meeting with a lawyer who suggested otherwise. The team is definitely feeling anxious to pick a project and with funding deadlines approaching we decided to set a deadline for next week.
In addition to literature review and HP, we continued our other work in graphic design for the education slides and software skill development.
May 25 - 29
We had a lot of long meetings this week but we finally picked a project!! After a few more HP meetings and internal meetings discussing feasibility of certain ideas we narrowed down to four to be presented as contenders for 2020 project.
After the continued research of this week and the various HP meetings we narrowed in on four ideas: Bacterial Cellulose Skin Care, Biomining/Bioleaching, Probiotic Gum for Dental Caries, and a sustainable GMO Yeast Supplement for Vitamin A deficiency. Each group presented in lengths the pros and cons of the projects, outlining the prospects for HP and dry lab, while also critically analyzing the feasibility for wet lab and the marketability. After this very long discussion we conducted a vote and decided that the GMO Yeast Supplement for Vitamin A Deficiency would be our project.
At the end of the week we started looking more into the wet lab components and how to do the genetic manipulation itself as this is a new chassis for us. Dry lab also brainstormed projects they could start working on for this and we had preliminary discussions regarding modelling possibilities. Our next goal is to get more HP meetings going, do some more research for the wet lab, and start focusing on other subgroups like JulyGEM, Funding, cGEM, Education, and the Faculty Talk.
OMEGA: June
Cellulase | Omega/thymol | Biosafety | Hardware | Software
June 1 - 4
We started reading papers about EPA and DHA production in microbial systems. We found EPA gene cluster but it was 38kb. We’re having difficulties looking for specific desaturase genes because papers do not include their accession numbers.
June 8 - 12
We found a paper outlining genes required for EPA production in S. cerevisiae and their accession codes. We started creating our R. glutinis-specific constructs in Benchling.
June 15 - 19
Had a meeting with Dr. Zaremberg about engineering R. toruloides and considerations we have to make. She didn’t raise any concerns about producing omega-3 fatty acids in the yeast, but told us it biotech industries have done great optimizations to make its production viable.
June 22 - 26
No omega-specific work was done this week.
June 29 - July 3
Started modelling the effects of Omega-3 biosynthesis in R. toruloides and its effects to the lipid bilayer. Looked at other omega-3 fatty acid expression such as ALA and DHA.
OMEGA: July
Cellulase | Omega/thymol | Biosafety | Hardware | Software
July 6 - 10
Setting up a meeting with Dr. Sanou Dia, a public health nutritionist to see if omegas have merit. We started looking for cheaper ways to characterize omega 3 production asides from gc/ms.
July 13 - 17
Looked at uses of R. toruloides in fortifying aquatic livestock with beta carotene and promoting omega-3 fatty acid production. Did more review on its potential use in sheep and cattle.
July 20 - 24
Looked at uses of R. toruloides in fortifying aquatic livestock with beta carotene and promoting omega-3 fatty acid production. Did more review on its potential use in sheep and cattle.
July 27 - 31
No omega-specific work was done this week.
OMEGA: August
Cellulase | Omega/thymol | Biosafety | Hardware | Software
August 3 - 7
We started working on protocols and their troubleshooting for EPA expression in R. toruloides.
August 10 - 14
No omega-specific work was done this week.
August 17 - 21
Protocols required were compiled. Our part design will be modelled after cellulase parts. Our team is now working on troubleshooting potential errors for omega 3 production
August 24 - 28
Due to our HP contacts’ feedback, we scrapped our omega-3 production subproject and worked on expression of a natural deworming agent instead. We started conducting literature review on how we might do this, and decided to express thymol as our anthelmintic agent.
August 31 - September 3
Talked to Dr. Paul Mains who works with drug resistance in C. elegans. He provided some insights on experimental design with our thymol project. We also ordered thymol for C. elegans testing. Found the genes responsible for thymol biosynthesis.
OMEGA: September
Cellulase | Omega/thymol | Biosafety | Hardware | Software
September 14 - 18
Designed the wetlab workflow for thymol production.
September 21 - 25
Continued working on designing the wetlab workflow for thymol production.
OMEGA: October
Cellulase | Omega/thymol | Biosafety | Hardware | Software
October 5 - 9
Wiki writing
October 12 - 16
Wiki writing
October 12 - 16
Wiki writing
BIOSAFETY: May
Cellulase | Omega/thymol | Biosafety | Hardware | Software
May 4 - 8
This was our first full week of iGEM Calgary 2020! We narrowed down the final project ideas to Biomining, Cow Herpes, Mycotoxins, and Mycorrhizae. Through information gathered from our literature reviews and Human Practices meetings, we also began to look into Johne’s Disease as a potential project area, but ultimately eliminated both Cow Herpes and Johne’s Disease. By the end of the week, we turned our attention only to Mycorrhizae, Mycotoxins, and Biomining.
With regards to mycotoxins, we began literature review to determine the need for and feasibility of a lactobacillus probiotic that contains degradation enzymes for major mycotoxins found in animal feed. In biomining, we originally had the idea to use a purified lanthanide binding protein, Lanmodulin, to bind the metals. However, following further literature review and Human Practices meetings, we decided that it is more feasible to express the protein on the cell surface of a genetically engineered microorganism (lower cost) or engineer our own binding tag/binding protein hybrid and display that on the cell surface.
Throughout the week, our dry lab members were hard at work brushing up on their software skills and brainstorming innovative hardware and software ideas for each potential project area. In addition, our team began working on graphic design for our educational tools! Finally, we ended our week with a virtual team building workshop, where we completed small group challenges and developed the 6 Rules of iGEM Calgary.
May 11 - 15
With our focus mainly on Biomining, Mycotoxins, and Mycorrhizae this week we went into full swing with HP meetings to narrow in on a project idea. We also had our first Synbiolympics event!
For Mycotoxins we had a meeting with a couple researchers from the University of Saskatchewan and a dairy cow veterinarian. We learned from our HP meetings that this problem may not be very relevant in Alberta and that there seem to already be effective solutions for it. Specifically, a company called Biomin has a lot of products and patents surrounding mycotoxins handling. We determined we need to do more searching to see if this project was worth it. Despite promising papers and HP meetings with a few professors, the Mycorrhizae project was unfortunately abandoned as the scope was determined to be too big and out of our capabilities. From the HP meetings last week Biomining had been having some patent troubles so we were trying to see if we could change the proteins enough to get around the patents. Bioleaching also became a new avenue to explore.
While also participating in literature review and HP, our dry lab members continued to learn new software programs and skills and they started a project called Bellatrix. A new idea was also proposed to look at DIY lab equipment.
With our main three project ideas from last week dwindling in support we decided to have a larger brainstorm session to look into new ideas. We had pitches relating to bacterial cellulose for skin care, miRNA or peptides for anti-inflammatory protection, probiotic gum to treat dental caries, silica nanoparticle-producing algae, and yeast supplements for vitamin A deficiency.
May 19 - 22
This week our team branched off into many little subgroups to focus on the various new ideas we pitched last week. In addition, we had our first education meeting to discuss our many ideas for education, and also started to get some other subgroups forming like JulyGEM and cGEM.
All of the subgroups had worked on fleshing out their ideas and doing some preliminary HP and patent analysis. However, we started making some larger team decisions on whether we should really allow patents to limit our ideas as we had a meeting with a lawyer who suggested otherwise. The team is definitely feeling anxious to pick a project and with funding deadlines approaching we decided to set a deadline for next week.
In addition to literature review and HP, we continued our other work in graphic design for the education slides and software skill development.
May 25 - 29
We had a lot of long meetings this week but we finally picked a project!! After a few more HP meetings and internal meetings discussing feasibility of certain ideas we narrowed down to four to be presented as contenders for 2020 project.
After the continued research of this week and the various HP meetings we narrowed in on four ideas: Bacterial Cellulose Skin Care, Biomining/Bioleaching, Probiotic Gum for Dental Caries, and a sustainable GMO Yeast Supplement for Vitamin A deficiency. Each group presented in lengths the pros and cons of the projects, outlining the prospects for HP and dry lab, while also critically analyzing the feasibility for wet lab and the marketability. After this very long discussion we conducted a vote and decided that the GMO Yeast Supplement for Vitamin A Deficiency would be our project.
At the end of the week we started looking more into the wet lab components and how to do the genetic manipulation itself as this is a new chassis for us. Dry lab also brainstormed projects they could start working on for this and we had preliminary discussions regarding modelling possibilities. Our next goal is to get more HP meetings going, do some more research for the wet lab, and start focusing on other subgroups like JulyGEM, Funding, cGEM, Education, and the Faculty Talk.
BIOSAFETY: June
Cellulase | Omega/thymol | Biosafety | Hardware | Software
June 1 - 4
This week was the official start of oOil project. We started looking into kill switches as a way of biocontainment for the genetically modified yeast. We soon realized that idea of using auxotrophy for biocontainment is not feasible or economical. Instead, we focused on genetic constructs that act as kill switches when they are induced by an environmental stimulus. We began to identify longevity as the biggest problem with these kill switches as they are not evolutionary stable. Toxin-antitoxin systems could be used to reduce the fitness cost of the kill switch. We then started looking into toxin-antitoxin systems that could be implemented in our kill switch. By next week we are hoping to have selected or short-listed some toxin-antitoxin systems that we can use.
June 8 - 12
We found some light-inducible systems that we could use for our kill switch. We initially thought of using a cas9 light-inducible system to target essential genes in R.T that do not have human orthologs. However, we soon realized that cas9 might not be the most viable option as it can be completed and take a long time to be effective. We will be looking into more direct toxic molecules to use in our kill switch.
June 22 - 26
This week we decided to Diana cas9 as a kill switch. After more discussion with Dr. Marija we figured that a cas9 system would required a lot of fine-tuning and is too time-consuming especially with all the other projects going on at the same time. We decided to go with something simpler. I started looking into spliceosome inhibitors as a way to kill the yeast. I found some molecules that effectively inhibit a crucial splicesome protein called SF3B1 but I later learned that these inhibitors are hard to synthesize biochemically and they are not effective against yeast SF3B1 protein. During a kill switch meeting, we figured that nucleases are currently our best choice of toxin. I started looking into blunt nucleases with short GC rich recognition sequences that could cut the Rhody T many times. I created a shortlist of promising restriction enzymes and screened the Rhody T genome for restriction sites to determine how prevalent each one was.
June 29 - July 3
Our kill switch system is also has many limitations as pointed out by our audience members which is something we need to look into. Next week we will be meeting with Robert Mayall to discuss or kill switch system further and gain some inspiration. I did briefly look into idea of using quorum sensing in our kill switch system. However, this concept seems way too complex and is not worth pursuing.
BIOSAFETY: July
Cellulase | Omega/thymol | Biosafety | Hardware | Software
July 6 - 10
Within the biosafety group we dina’d the idea of using light inducible system for our kill switch following our meeting with Robert. We instead decided to focus on auxotrophies. In particular, making a syntrophic community of yeast that have complimentary auxotrohpies. We conducted a lot of lit review on cooperative auxotrophic yeast and bacteria communities. We also met with Marija to discuss our new idea. She liked our idea but mentioned that it would require a lot of work. She suggested we start working on S.C first before working with Rhody T. We also met with dry lab to discuss modeling given the immense modeling potential that this project has. We still need to conduct more research in this area as we still know a little and require more lit review to make informed decisions.
July 13 - 17
This week we dove deeper into our new auxotrophy co-culturing idea. We found literature that have done auxotrophic co-culturing with S.C with relative levels of success. We looked further into amino acid overproduction. We had a meeting with Dr. Chua to discuss our new idea with him. He liked our idea but cautioned us that it could be difficult to pull off. He suggested that we start working with two strains of yeast and add third and fourth if successful. He also pointed to several other things that we need to consider such as chances of contamination, duration of co-metabolism, balance of nutrients and potential toxic byproducts produced by the strains. Finally, he gave us some people to contact such as Dr. Zaramberg , Rho and Dunfield. Our plan is to meet with Marija next week and the rest of the wet lab to discuss our idea one last time. If we decide to double down on this then we would start working on the experimental design.
BIOSAFETY: August
Cellulase | Omega/thymol | Biosafety | Hardware | Software
August 17 - 21
This week, we started looking into Yarrowia lipolytica as an alternative chassis to Rhodotorula toruloides. Members of our subgroup specifically looked into existing/possible auxotrophic or overproducing strains, sequences for beta-carotene production, and effective promoter and terminator sequences.
August 24 - 28
Presented our biocontainment project idea at the summer symposium at our university. Was well received. Met with Dr. Rodrigo, who had misgivings about our strategy using co-culturing due to the prevalence of amino acids in the natural environment. Decided to contact Dr. Robert Mayall to gain some advice on how to move forward.
August 31 - September 1
Week off. Met with Robert Mayall again who reassured us of our biocontainment idea. He said that we could conduct experiments using soil and lake samples to show that our yeast could not grow outside of the bioreactor conditions. Told us that the concentration of amino acids in outside environment should not be high enough to pose a problem for our biocontainment strategy.
BIOSAFETY: September
Cellulase | Omega/thymol | Biosafety | Hardware | Software
September 14 - 18
Monday (Sept. 14): Arshia and Nimaya made amino acid solutions for Leu and Met for the SD Media preparation, which we plan to do once all of the amino acids come in. We also made plain YPD media.
Tuesday (Sept. 15): We acquired plasmids, S. cerevisiae, and Y. lipolytica from the Zaremberg lab. Tian made some Y. lipolytica overnights for us to use.
Wednesday (Sept. 16): Anika poured plain YPD plates from the media we made on Monday and also made more CaCl2 for use in bacterial transformations. Glycerol stocks on Y, lipolytica and the S. cerevisiae W303 strain were made for later use. Nimaya and Arshia also miniprepped both of the plasmids containing Leu2 and Trp1 from bacteria.
Friday (Sept. 18): Today we finished miniprepping the Leu and Trp plasmids stored in E. coli and nanodropped. The concentration and purity of the miniprep samples were written down and the best miniprep was chosen to be used later for yeast transformation of wildtype W303 strain. We also remade glycerol stocks of both wildtype W303 and Yaroowia strain from plates because last time the glycerol stocks were made, the overnights were not grown at the right temperature.
September 21 - 25
Monday (Sept 21): Today we streak-plated the both engineered W303 strains (the Leu auxotroph and Trp auxotroph) on YPD media to observe colony morphology and growth on YPD media. Besides that we didn’t do much grim reaper-related work today as we are still waiting on tryptophan to arrive. Once we get tryptophan, we would have all the necessary reagents and amino acids needed to make all the required media for grim reaper’s experiments.
Wednesday (Sept 23): We prepared amino acid and nucleotide solutions to be autoclaved, as this is the first step before we can make the appropriate SD media. In total, we made 6 solutions--these will be sent to autoclaving. Once we get them back from autoclaving, we can prepare our SD media/agar. Today, we also did our first attempt at a yeast transformation, as we attempted to transform the wild type W303 S. cerevisiae strain with a miniprepped Leu2 plasmid and a control. We used stationary phase culture (overnights) for this protocol. Next time we do the transformation, we will use mid-log phase culture. We finished the transformation, but were not able to get to plating--we will do this next time.
Friday (Sept 25): No lab today because of COVID precautions. We were planning to pour the SD Leu- plates that were autoclaved that morning. This was instead done by Deirdre and the plates were stored in the fridge. On Monday we would have to correctly label the plates and then plate our yeast transformation on the SD Leu- plates.
September 28 - October 2
Monday (Sept. 28): We were originally planning to perform more transformations of the S. cerevisiae W303 strain with mid-log phase cultures expressing the Leu2 plasmid and a control. However, there were no mid-log cultures prepared. So instead, we spent the lab day preparing SD media and agar, specifically SD-Trp, SD-all amino acids, and SD-Leu--these will be sent to autoclaving. Additionally, we plated the W303 Leu2 transformations from last week onto YPD and SD-Leu plates. We plated on YPD plates to verify if the cells are alive, as YPD contains all the nutrients required for their growth. Growth on SD-Leu verifies that the transformation is successful. Finally, we streaked our engineered S. cerevisiae strain (YMM65, the Leu auxotroph) onto SD-Leu plate to observe its growth.
Wednesday (Sept. 30): We noticed that our plates from last day seemed to have contaminated growth on them, so we’ll have to redo those plates on Friday. Today, we restreaked the original plate that had E. coli expressing Leu2 and Trp1 plasmids. We also streaked the S. cerevisiae W303 auxotrophic strain on our three different plates (SD Leu-, SD Trp-, and SD fully supplemented) in order to confirm auxotrophy and ensure that the media isn’t supplemented with the amino acids. We also did mid-log W303 yeast transformations with the Leu and Trp plasmids, as well as a control, and plated all three. Tian had prepared the mid-log cultures for us earlier in the day. We poured plates for SD Leu- and SD fully supplemented media.
Friday (Oct 2): Both of the yeast transformations (Leu2 and Trp1 expression) done on Wednesday were successful! As a result, we were able to start yeast characterization experiments today. Today, we started experiments to determine the standard curve of OD600 vs concentration of leucine for the auxotrophic W303 strain. The purpose of this experiment is to set up standards for characterizing the overproduction of leucine by the engineered yeast cells. A series of SD media supplemented with different concentrations of leucine (0, 3, 6, 9, 12, 15mg/L) were set up. One control (no yeast cells) and three replicates for all amino acid concentrations containing the same amount of yeast cells were loaded into a 96-well microplate reader and OD600 was measured every 15 minutes over the course of 24 hours. The data from the microplate reader will be analyzed, most likely on Monday.
BIOSAFETY: October
Cellulase | Omega/thymol | Biosafety | Hardware | Software
October 5 - 9
Monday (Oct. 5):Today, we made liquid SD-Trp- media so we can run the same experiment we did on Friday but to make a standard curve for tryptophan concentrations. This experiment will most likely be run on Wednesday or Friday of this week. We also made glycerol stocks of the Trp-producing and Leu-producing transformed W303 S. cerevisiae strains. We also started analyzing the plate reader data from Friday’s experiments to create a standard curve of OD600 vs leucine concentrations.
Wednesday (Oct. 7): We discovered that the 1% Trp solutions we had made a few weeks ago were no longer usable, as Trp degrades when exposed to light and heat. Therefore, we could not do the plate reader experiments to create our standard curve for the Trp concentrations. We plan to filter-sterilize to make a new stock of 1% Trp solution on Friday. However, we were able to do a plate reader experiment to measure the amino acid concentrations produced by the ymm60 engineered strain (Leucine overproducing strain). This consisted of first growing mid-log cultures and overnights of the ymm60 strain in SD-Leu media (this was done for us already by our TA). Then, we used centrifugation and a nylon membrane to filter out the cells from the media, so that we only had the supernatant containing the leucine that the strain should have overproduced. We then completed a similar protocol to Friday wherein we used the cells from mid-log cultures of W303 to inoculate samples of the ymm60 culture supernatant. We had a total of 8 samples with 200uL in each of the wells in the plate reader. The plate reader was run under the same conditions as last day. These results will be analyzed in the next few days.
Friday (Oct. 9):Today, we filter-sterilized a new stock of 1% Trp solution. Using this Trp solution, we were able to do a plate reader experiment to determine the standard curve of yeast growth in Trp-supplemented solution at the concentrations of 0mg/L, 3mg/L, 6mg/L, 9mg/L, 12mg/L, and 15mg/L. We also grew overnight and midlog cultures of ymm65 (Trp-overproducing) in SD-Trp- media. We then started another plate reader experiment of mid-log W303 cells in the SD-Trp media produced by the ymm65 cells to characterize Trp production by the strain. Arshia also started a cell counting experiment to determine the standard curve for OD600 vs cells.
October 12 - 16
Monday (Oct. 12):No lab today, Thanksgiving long weekend.
Wednesday (Oct. 14): We completed the amino acid plate reader experiments for mid-logs and overnights of W303 Leu transformed cells and Trp transformed cells. The protocol for these experiments were the same as those followed last day for the ymm65 strain plate reader experiment.
Friday (Oct. 16): We completed the environmental plate reader experiments today using autoclaved and non-autoclaved Bow River, and soil samples. Essentially, we grew our overproducing engineered strains, ymm60 and ymm65, in these different samples as monocultures, in a 96-well plate to measure the OD600 values over time (for a total of 60hrs). We also included positive controls as well. The goal is to see if any growth of cells takes place, as this will indicate that the yeast will be able to grow in the environment if an accidental release event were to occur. These results will be analyzed next day. Additionally, we made SD-Trp-Leu media of varying amino acid concentrations to prepare for the co-culturing experiments that will be taking place next week.
October 19 - 23
Monday (Oct. 19): Today we re-filtered the autoclaved and non-autoclaved dirt solutions in order to remove more soil particles in order to prepare for a repeat of the OD600 plate reader experiments this week for the environmental tests. Additionally, the plate reader results from the amino acid overproduction bioassay was analyzed. Based on the results we decided to make cultures of the ymm60, ymm65, Leu-transformed, Trp-transformed yeast cells to create a 48hr and two 96 hr overnight cultures that will be used to rerun the plate reader experiments. We are hoping that more amino acids will be released into the media after the cells have grown for a longer period of time. These plate reader experiments will be run Wed. and Friday of this week.
Wednesday (Oct. 21): We planned to repeat the amino acid characterization experiments from last week but with 24 hr and 48 hr cultures. However, there was an issue with our shaker, and most of our overnight cultures were compromised. Thus, we just re-did the plate reader experiments for the Leu and Trp standard curves, and repeated the environmental sample tests with ymm65 strain. The Trp standard curve included smaller increments from 0-6 mg/L instead of from 0-15 mg/L that we completed last time.
Friday (Oct. 23): We received more bad news. The plate reader experiments from last day were prematurely ended, causing us to lose our results. Thus, today we repeated the experiments that were meant to happen on Wednesday: we repeated the amino acid plate reader experiments using 24 hr and 48 hr cultures. We also repeated the Leu and Trp standard curves. We also made co-cultures with different SD media that had varying concentrations of Leu and Trp, and used a fluorescent plate reader to make measurements. We will analyze these results in the next few days.
October 26 - 27
WIKI WRITING
CELLULASE: May
Cellulase | Omega/thymol | Biosafety | Hardware | Software
May 4 - 8
This was our first full week of iGEM Calgary 2020! We narrowed down the final project ideas to Biomining, Cow Herpes, Mycotoxins, and Mycorrhizae. Through information gathered from our literature reviews and Human Practices meetings, we also began to look into Johne’s Disease as a potential project area, but ultimately eliminated both Cow Herpes and Johne’s Disease. By the end of the week, we turned our attention only to Mycorrhizae, Mycotoxins, and Biomining.
With regards to mycotoxins, we began literature review to determine the need for and feasibility of a lactobacillus probiotic that contains degradation enzymes for major mycotoxins found in animal feed. In biomining, we originally had the idea to use a purified lanthanide binding protein, Lanmodulin, to bind the metals. However, following further literature review and Human Practices meetings, we decided that it is more feasible to express the protein on the cell surface of a genetically engineered microorganism (lower cost) or engineer our own binding tag/binding protein hybrid and display that on the cell surface.
Throughout the week, our dry lab members were hard at work brushing up on their software skills and brainstorming innovative hardware and software ideas for each potential project area. In addition, our team began working on graphic design for our educational tools! Finally, we ended our week with a virtual team building workshop, where we completed small group challenges and developed the 6 Rules of iGEM Calgary.
May 11 - 15
With our focus mainly on Biomining, Mycotoxins, and Mycorrhizae this week we went into full swing with HP meetings to narrow in on a project idea. We also had our first Synbiolympics event!
For Mycotoxins we had a meeting with a couple researchers from the University of Saskatchewan and a dairy cow veterinarian. We learned from our HP meetings that this problem may not be very relevant in Alberta and that there seem to already be effective solutions for it. Specifically, a company called Biomin has a lot of products and patents surrounding mycotoxins handling. We determined we need to do more searching to see if this project was worth it. Despite promising papers and HP meetings with a few professors, the Mycorrhizae project was unfortunately abandoned as the scope was determined to be too big and out of our capabilities. From the HP meetings last week Biomining had been having some patent troubles so we were trying to see if we could change the proteins enough to get around the patents. Bioleaching also became a new avenue to explore.
While also participating in literature review and HP, our dry lab members continued to learn new software programs and skills and they started a project called Bellatrix. A new idea was also proposed to look at DIY lab equipment.
With our main three project ideas from last week dwindling in support we decided to have a larger brainstorm session to look into new ideas. We had pitches relating to bacterial cellulose for skin care, miRNA or peptides for anti-inflammatory protection, probiotic gum to treat dental caries, silica nanoparticle-producing algae, and yeast supplements for vitamin A deficiency.
May 19 - 22
This week our team branched off into many little subgroups to focus on the various new ideas we pitched last week. In addition, we had our first education meeting to discuss our many ideas for education, and also started to get some other subgroups forming like JulyGEM and cGEM.
All of the subgroups had worked on fleshing out their ideas and doing some preliminary HP and patent analysis. However, we started making some larger team decisions on whether we should really allow patents to limit our ideas as we had a meeting with a lawyer who suggested otherwise. The team is definitely feeling anxious to pick a project and with funding deadlines approaching we decided to set a deadline for next week.
In addition to literature review and HP, we continued our other work in graphic design for the education slides and software skill development.
May 25 - 29
We had a lot of long meetings this week but we finally picked a project!! After a few more HP meetings and internal meetings discussing feasibility of certain ideas we narrowed down to four to be presented as contenders for 2020 project.
After the continued research of this week and the various HP meetings we narrowed in on four ideas: Bacterial Cellulose Skin Care, Biomining/Bioleaching, Probiotic Gum for Dental Caries, and a sustainable GMO Yeast Supplement for Vitamin A deficiency. Each group presented in lengths the pros and cons of the projects, outlining the prospects for HP and dry lab, while also critically analyzing the feasibility for wet lab and the marketability. After this very long discussion we conducted a vote and decided that the GMO Yeast Supplement for Vitamin A Deficiency would be our project.
At the end of the week we started looking more into the wet lab components and how to do the genetic manipulation itself as this is a new chassis for us. Dry lab also brainstormed projects they could start working on for this and we had preliminary discussions regarding modelling possibilities. Our next goal is to get more HP meetings going, do some more research for the wet lab, and start focusing on other subgroups like JulyGEM, Funding, cGEM, Education, and the Faculty Talk.
CELLULASE: June
Cellulase | Omega/thymol | Biosafety | Hardware | Software
June 1 - 4
We finally started doing some wet-lab specific research this week! At the beginning of the week we outlined what was necessary for us to research as a group and developed research responsibilities for each member.
June 8 - 12
We researched different chassis, particularly R. glutinis and R. toruloides and determined the 3 classes of cellulase enzymes that the yeast must produce. We found six total enzymes that we could potentially integrate into the yeast, although we are looking to cut this number to 3. After further investigation into the BGS cellulase enzyme, we realized that we would need to do kinetics modelling to ensure that glucose does not hinder its pathway.
June 15 - 19
This week, Dr. Mayi and Marija created a yeast biotech lecture for us. We also met with Marija to discuss potential homology sequences that we would need to target. Additionally, we worked on narrowing the list of cellulases for integration into our yeast.
June 22 - 26
This week, we finalized our cellulase genes, and met with the modelling team regarding modelling of some of the cellulase enzymes for better function in R. toruloides.
June 29 - July 3
This week, we started a document on the potential homology sequences we could target. Our potential options are looking at recombining with non-essential genes, looking at the intergenic locations adjacent to non-essential genes, and the Ku70/80 complex. We also created a new communication channel on Slack for cellulase-related work.
CELLULASE: July
Cellulase | Omega/thymol | Biosafety | Hardware | Software
July 6 - 10
This week, we designed primers for the cellulase constructs and resolved any hairpin and stem loop issues, as well as created a guide to primer design for future new members. We also completed a wet lab abstract for submission to the iGEM Maastricht journal. We started looking into potential glucose tests to use in the lab, and reached out to Marija and Chris for guidance. We also asked them where to find cellulose, cellobiose, and other intermediates, as well as Gibson reagents. Finally, we looked at a R. glutinis paper to see which CBHs were used, found the FASTA sequences for these proteins in UniProt, and codon optimized these proteins.
July 13 - 17
We received notice that we would be allowed back in the lab in the upcoming fall, although our hours would be rigid and restricted to 6 hours. We also researched different resistance genes, and ultimately selected bleomycin (also known as Zeocin) resistance because it is the smallest resistance gene (372bp) and has been used multiple times.
July 20 - 24
All wet lab members worked updating and documenting their lab safety training to prepare for the upcoming fall in the lab. We also completed more work on the troubleshooting document.
July 27 - 31
This week, we had a meeting to organize research of protocols and troubleshooting to avoid redundancy across subgroups, and completed more work on troubleshooting document. We also began preparing experiments to test the full GPD, half GPD, and full LPD1 promoters.
CELLULASE: August
Cellulase | Omega/thymol | Biosafety | Hardware | Software
August 3 - 7
This week, constructs for promoter testing were completed. Everyone checked them before ordering. We also designed constructs to include “Gibson to Yeast Homology” sites for attachment of homologous sequences for R. toruloides, should we decide to do so in the future. Finally, we completed more work on troubleshooting document
August 10 - 14
We worked extensively on Benchling to create our Gibson Assembly constructs and reduce their complexity scores. Reducing complexity scores was a significant undertaking, due to the GC richness of R. toruloides. Finally, we completed more work on troubleshooting document
August 17 - 21
This week, we started looking into Yarrowia lipolytica as an alternative chassis to Rhodotorula toruloides. Members of our subgroup specifically looked into past engineering of beta-carotene production in Y. lipolytica, levels of beta-carotene produced, and growth requirements. We also began searching for protocols, such as chemically competent preparation, transformations, and protein extraction. Finally, we began compiling a list of all the iGEM parts that have been made for Yarrowia.
During our search, we found a paper on the engineering and optimization of beta-carotene production in Y. lipolytica, and contacted the authors for a potential meeting in order to better understand the benefits of Y. lipolytica as a chassis for our project. We also found that Y. lipolytica has been better characterized than R. toruloides, and that protocols from S. cerevisiae are easily transferable to Y. lipolytica. Furthermore, Y. lipolytica has been approved for consumption in killed form by the European Food Safety Authority, and is Generally Recognized As Safe (GRAS) by the FDA.
We reconvened with other wet lab subgroups to discuss our findings, where we decided a chassis pivot to Y. lipolytica was worth pursuing. We then reached out to the whole team, where the dry lab agreed that a pivot would be beneficial. After careful deliberation, we decided to make the jump - Y. lipolytica was our new chassis!
August 24 - 28
This week, we had a meeting with Dr. Rodrigo Ledesma-Amaro, a research lead at the interface of synthetic biology at the Imperial College London. As he has authored many papers doing synthetic biology on Y. lipolytica, including engineering and optimizing beta-carotene production. Fortunately, he kindly agreed to send us a strain of Y. lipolytica that produces beta-carotene production. This will speed up our own research timeline, as we now only have to optimize beta-carotene production in Y. lipolytica.
Due to our pivot in chassis, we had to contact IDT regarding a refund for our order. A massive thank-you to Tami from IDT customer support who gave us a full refund, restoring our full IDT allowance! We went ahead and re-made all of our constructs on Benchling for Yarrowia after researching good promoters, terminators, and signal peptides. We also referred to some previous papers that have introduced cellulases to Yarrowia.
We also presented our cellulase work at the Summer Student Symposium at the University of Calgary, which was well-received.
August 31 - September 3
Although this was our week off, we worked extensively on Benchling to reduce complexity scores and finalize our cellulase construct design in Y. lipolytica. After thoroughly checking our constructs, we ordered them from IDT and Twist. Yay! We also created a master excel sheet for Chris to place orders for lab reagents.
CELLULASE: September
Cellulase | Omega/thymol | Biosafety | Hardware | Software
September 7 - 11
After 4 long months we finally got into the lab!
Tuesday (Sept 8): Planning of wet lab experiments for the next day.
Wednesday (Sept 9): Today was our first day in the lab! After a short safety spiel from Chris and Tian, we went ahead and started our work. We had Tian make overnight cultures of DH5a E. coli the night before, so Chris came in a couple hours before us and started the subcultures for making chemically competent cells. We continued subculturing until all four cultures had an OD600 of 0.3-0.4 (took full two hours). After which, we transferred the cultures into pre-chilled 50mL falcon tubes, spun down the culture, discarded the supernatant, and froze the pellets. We will resume the rest of chemical competent cell preparation on Friday.
Meanwhile, a few of us prepared 14 cPCR mastermixes and put them back in the freezer for use. We also had Tian make an extra DH5a overnight to make a new glycerol stock of DH5a to store in the -80 freezer. We also prepared more 50X TAE buffer and 1X TAE buffer from that. We also planned to do media prep, but unfortunately the autoclave closed before we could send down the media, so we will do that on Friday instead.
Thursday (Sept 10): Tian set up four BL21 empty pSB1A3 overnight cultures so we can miniprep the pSB1A3 DNA on Friday.
Friday (Sept 11): Today we finished our work on the competent cell prep and aliquots of competent cells were stored in the -80 degrees refrigerator. The competent cells will be transformed next week to ensure their competency. The appropriate antibiotic was added to the autoclaved media that was prepared earlier by Chris and Tian. The media was then poured into properly labeled plates and stored in the refrigerator after agar had solidified. Some of us also practiced miniprep on empty DH5a vectors and measured the concentration and purity of our miniprep using NanoDrop. We were planning to make a list of the new reagents, but unfortunately, we ran out of time, so we will have to do that first thing next week.
September 14 - 18
Monday (Sept 14): Some of us performed a practice digest of 2019’s RFP Flipper in pSB1A3 using KpnI and then performed a practice gel electrophoresis with that digest. Due to lack of DNA, the finished gel was very faint, so we used the gel to practice cutting the gel and isolating the bands. stored in the (which) refrigerator, and results will be analyzed on Wednesday. During the cooling and incubation periods for the gel electrophoresis, we planned our poster for the upcoming Concordia Mini Jamboree.
Wednesday (Sept. 16): No growth was observed from the reincubated test transformations with the chemically competent cells made the week before, so new chemically competent cells were made. For practice and a control, we transformed RFP into the old (from iGEM 2019) and new (last week’s) chemically competent cells to ensure the new batch was faulty. The DNA parts that arrived yesterday were resuspended in TE buffer to a concentration of 100ng/uL and will need to be aliquoted to prevent stock contamination.
Friday (Sept 18): Today was a very exciting day as we got to work with our IDT DNA. We received 4ug of dried plasmid DNA of TrCBHII, WtpTEF1 promoter, and NcCBHI, that was resuspended in TE buffer on Wednesday. We did transformations with 50ng and 100ng of DNA for each of the three plasmids. We plated on LB-amp plates and left them on the countertop (with parafilm) over the weekend. Additionally, we made the two mastermixes to digest pSB1A3 next week for when our Twist DNA arrives. Our IDT DNA is already in plasmids, but our Twist DNA was ordered as gene fragments, therefore we have to digest and ligate them into pSB1A3 first. We made two mastermixes one with EcoRI + PstI and the other with XbaI + PstI.
September 21 - 25
Monday (Sept 21): We checked our transformations from Friday and to our surprise had lawn growth. We suspect this might have been because we left them to grow over the weekend for too long. Regardless, we decided to replate the transformations (done by Anika and Arshia), and also made a streak plate from a glycerol stock with pSB1A3 in it just to verify that our ampicillin plates were good. In the meantime, Allison did a cPCR of the transformation plates with colonies she could find on the plates or otherwise took a stab from the edge of the lawn. We put the plate into the thermocycler and set the PCR conditions, Tian will remove it and put it in the fridge tomorrow morning for us to do gel electrophoresis. All the re-plated transformations, the streak plate, and the masterplates were put in the incubator.
We also conducted a few dig-ligs today. So we took the two mastermixes from Friday and digested some pSB1A3. We also got the Twist DNA today, so we resuspended that dried DNA in TE buffer and then digested those gene fragments. The two gene fragments we received from Tist were BGS (part 2) and BGS (part 3), and for each we tried a 4:1 ligation and a 1:1 ligation. Once the digests were finished, the ligation reactions were set up and left on the counter. Tian will put them in the freezer tomorrow morning for us to transform on Wednesday.
Finally, we received the Nourseothricin construct today, so we resuspended it in TE buffer and put it in the freezer. We will transform it on Wednesday.
Tuesday (Sept 22): Tian is going to come in to check some things which will dictate our plans on Wednesday:
Check Anika and Arshia’s test transformations on Chlo plates
Red colony growth= They worked!
No growth= Put them back in incubator and make 4 new DH4a overnights and shincubate at 28ºC NOT 37ºC
Check pSB1A3 glycerol stock streak plate
Proper growth= ampicillin plates are good
Bad growth= ampicillin plates might be messed up and we need to remake them
Put cPCR 96-well plate in the fridge
Put ligation products in the small -20 freezer. Includes…
BGS part 2 4:1
BGS part 2 1:1
BGS part 3 4:1
BGS part 3 1:1
Check re-plated transformations for proper colony growth
Proper growth= Parafilm and put in fridge
No growth= Leave in incubator
Check cPCR masterplates
Proper growth= Make 2mL O/N cultures with LB-amp- make three O/Ns per gene (not per plate- should be 9 O/Ns total)
No growth= leave in incubator
Wednesday (Sept 23) We ran the cPCR products on a gel and miniprepped the overnights made from the master plates. We also transformed the nourseothricin construct and the four ligation products (BGS part 2 4:1, BGS part 2 1:1, BGS part 3 4:1, BGS part 3 1:1)
CELLULASE: October
Cellulase | Omega/thymol | Biosafety | Hardware | Software
October 5 - 9
Monday (Oct 5): We planned to complete a cPCR of our new BGS2 and BGS3 ligation colonies, but there was insufficient growth of the colonies. Instead, we prepared cPCR mastermix. We also nanodropped the minipreps of our Penny colonies and prepared them for sequencing. The BGS3 4:1, nourseothricin, nourseothricin plate 2, and penny minipreps were discarded as the concentrations were extremely low. We also redid the transformations of SEGI8, Nourseothricin, WtpTEF, and hrGFP bronze medal with 50ng DNA.
Tuesday (Oct 6): Tian sent the BGS3 1:1, TrEGII, and TrCBHII minipreps for sequencing, as those minipreps were good.
Friday (Oct 9): We redid the digestions/ligations of BGS2 and BGS3 as 1:1 and 3:1 into pSB1A3. We also conducted cPCR of the wtpTEF promoter, Nourseothricin, and Penny transformations from Wednesday. In doing so we had to make more cPCR mastermix, but it seems we’ve run out of taq polymerase! Uh oh! We also ran a gel of the cPCR products from Wednesday, unfortunately even our positive control looked strange as it seemed all the DNA was just stuck in the well and there was no plasmid DNA. We’re not sure why this is happening- perhaps the cPCR parameters aren’t good, the mastermix is messed up, or the primers aren’t good. The rest of the lanes on the gel gave no bands. We also replanted the transformations from Wednesday on carbenicillin plates to see if the lawn growth would persist, and left them at room temperature over the weekend.
We also checked the Yarrowia growth experiments, shook the cultures that needed to be shook, and then took the OD600 value.
October 12 - 16
Monday (Oct 12): Thanksgiving! We took the day off lab work to be with our families.
Wednesday (Oct 14) We performed gel electrophoresis on the wtpTEF promoter, Nourseothricin, and Penny cPCRs from Friday. Unfortunately, bands failed to appear on all three. We also performed transformations of the ligation products from Friday (BGS2 and BGS3 as 1:1 and 3:1 into pSB1A3)
Friday (Oct 16) We did cPCR on BGS 2 and BGS 3, and worked on the wiki.
October 19 - 23
Due to the approaching wiki freeze, we set aside DNA experiments and focussed on a variety of Yarrowia growth assays.
On Monday, we made DNS reagent, and set up an experiment determining the rate of breakdown of pure cellulose by a commercial cellulase mix. We also set up cultures of Yarrowia on pure glucose and water to measure their growth rate, and growth rate with varying oxygenation and length of heat shock.
On Wednesday, we collected those results from monday using spectroscopy. We also set up cultures of yarrowia with various sources of cellulose and cellulase enzyme added.
On Thursday and Friday, we measured the optical density of those cultures again to determine their growth rate.
October 26 - 27
Same as Thursday and Friday, we once again measured the optical density of the cellulose and cultures again to determine their growth rate
WIKI CONTENT WRITING!!
