Team:Consort Alberta/notebook

Our Notebook

   Introduction      Meetings   

Lab Work     Workshops

Introduction...

     The following notes are of our daily log book that was kept up throughout the year - the day to day activities, challenges, and solutions we come across while having our meetings or doing our lab work. Our lab work notes include the set protocols that we did and when, how, and why we did them. All of these notes are of all team member's work from several different points in time, combined together to give a full scope of what we did this year. Within the meeting notes are references to our lab procedures, which are listed below the meetings in detail of what we did, how we did it, and some photos too. The three workshops we went to are also described.

Meetings...

    September 16th: 3:30-4:15… Met the team from last year and the new member. We discussed the project idea that is continuing from last year's group (See last year's wiki for more information), and what synthetic biology and genetic engineering mean. We looked at a video about synthetic biology and a few other sites. Last year's Consort iGEM Team webpage was reviewed, as well as some of the other competition teams, and we looked through some of their wikis.

    September 30th: 3:30-4:30… We are getting a new Thermo-Cycler (PCR). The one we're getting is the BioRad PTC 200 (with 96 wells, like the old one, and has a heated lid). Along with new micro-pipettes (designed to measure things out in microliters). We further discussed other lab equipment that we may have to purchase. Looked over the Central Dogma of Biology, along with how different kinds of lab equipment work.

    October 7th: 3:30-4:15… Checked out other wikis for ideas for ours, and saved some links that we could refer back to and look at for inspiration.

    October 21st: 3:30-4:00… We are planning on doing an iGEM presentation to the Consort Lions Club on November 21st to get financial and community support. We discussed who else we could talk to about support and sponsorship too, and came up with a short list of people or businesses we could contact.

    November 18th: 3:30-5:00… We looked at the iGEM poster and determined who we need to contact for the biobricks and supplies we need for our project. The new students also looked at the process of DNA systems and about ribosomes with RNA.

    November 25th: 3:30-4:30… Darren and Sharon from the Consort Lion's Club presented our grant to us on behalf of the Consort Lions, as well as Sharon presented a second cheque on behalf of Ronwood Enterprises. On another topic, if things go well, we'll be submitting 2 biobricks to the registry at the end of the year.

    December 2nd: 3:30-4:00… A snow day at school, so a few iGEM students hung out in the science lab and began planning our wiki based on what we want, what we saw that was cool on other wikis, and what we needed for requirements. New member to the team: Alex Coulton joined late, and so we updated him on all the science and details. We are now a team of 8 students.

    January 6th: 3:30-4:00… We received an amazing grant from Alberta Innovates who was impressed with our application. We each will kick in a potential $200 for the iGEM trip, and we are each responsible for meals and souvenirs. Today we discussed what are website will look like - Nikayla is going to take candid photos for alongside the bios within the next meeting or two, and with the help of Kris and Austin will work on the technical side of the site. We're going to write the bios so they are ready for the next meeting. Next day at lunch: determined about 200 words for the bio, including favourite Canadian stereotype. We're also going to do a "very Canadian" photo at lunch depicting a barbeque, shorts and t-shirts, and maple syrup… in the snow.

    January 13th: 3:30-5:30… Did our photo today… it was… cold. We are going to have a workshop on February the 8th at the lab here at the school. Today we compared resources and website uploading options for the Wiki. We might go with OpenBexi. Going to come up with a shopping list for any supplies and such we need for the web lab.

    January 27th: 3:30-4:00… Workshop in Consort planning in progress - date is being shuffled around… not sure when or how it's going to work yet?

    February 10th: 3:30-4:30… Confirmed workshop date and time: February 23rd. Two people from the U of C are coming, and we are going to go over most of the procedures that we will be doing specifically to our needs. We tried downloading OpenBEXI to use on our website, but it's wonky and didn't work… so Kris contacted a student at Lethbridge to see what they used, and they used Microsoft Dreamspark - we'll try that out.

    February 23rd: We had our first Consort iGEM Workshop of the year with David Lloyd and Himika Dastidar from 8:30-5:00. (See this tab for more information)

    March 3rd: 3:30-4:00… Mr. Bourassa was gone, but we got all of our pages for our wiki drawn out as well as our main idea of our team descriptions and project info done. We hope to start experimenting with the website program soon!

    March 24th: 3:30-5:00… Discussed more fund raising, and got a few replies back from the letters we sent out. Any new modelling and software ideas are due for this Wednesday. Protocols to follow once we get our DNA… A meeting to follow on Wednesday and Friday at lunch.

    April 14th: 3:30-5:00… Fund raising follow-up - we received gracious grants from the Veteran Lions Club and Under Pressure. Sam conducted an interview with T&E Pumps for the Human Practices portion of our website. We also put Carla's Stringari as a credits for the logo and website. Alberta Innovates credits too, and we forwarded our articles to their contact as well. We also need to pick up supplies in Calgary! Once we get them, we should be able to start our plasmid switches and we need to schedule times and dates for our lab work. For our flights for the Jamboree - need names for everyone as they are on the passport plus our birthday.

    April 27th: We had our second workshop of the year, with Emily Hicks and Robert Mayall from 8:30-5:00. (See this tab for more information)

    April 28th: 3:30-5:00… More fundraiser follow-up and general part ordering and organization discussions. Website is well in progress, and a new modelling software is downloaded and begun to be experimented with.

    May 5th: 3:30-4:30… We will get some supplies in later this week so we organized the cabinets. We returned our incubator to the Kindergartens too. Arranging to go to a workshop in Calgary at the end of the month, and we'll see how that will go.

    May 12th: 3:30-4:30… On May 8th Kris happened to fix the autoclave… then we ran into some… uh… issues with the apparently heat-resistant bottles that we put into the sterilizing machine… they kinda melted, but they're sterilized! Website is in further progress, other teams are starting to do theirs a little too, but we are overall ahead of the game. Workshop on Saturday May 24th, and we will leave right after school to head to Calgary. We booked our geekStarter tickets and got them ready too.

    May 19th: 3:30-4:30… General meeting today; getting ready to make some LB Broth and going over procedures. For making the LB Broth, SOC and SOB media, see the lab work for more information.

    May 20th: 3:30-4:30… Made LB Broth with no complications. Also worked on modelling and website afterwards until 6:00. The beginning phases of mathematical modelling are appearing after the extra help from Robert Mayall.

    May 21st: 3:30-5:00… Took LB Broth out of the autoclave today. Before dealing with the more serious synthesized DNA, we are following our 3A Assembly Kit and Transformation Efficiency Kit from the iGEM Headquarters, which outline basic procedures that we'll use with our synthesized DNA later on. This book will take us through step by step and show us how to, in the end result, make E. coli cells produce a Red Fluorescent Protein that can directly tell us whether or not we did the procedure properly, as it has a strong reporter. Did some agar plates (SOB) and we are growing cultures overnight on the shaker table. Made 3 plates - two from the 2014 distribution and one from the 2013. For making the Agar Plates, see the lab work for more information.

    May 26th: 3:30-6:00… Today we made more plates and did agar stabs from the Kit and grew our E. coli. We did 6 agar stabs on plates, and got together the materials and procedures that we need for tomorrow. We've planned a mini-prep for Wednesday. Also worked on modelling afterwards. To look at our procedure, see the lab work for more information.

    May 29th: 3:30-4:30… We used a spectrophotometer to get an optimal density reading on our culture tubes. To look at our results, see the lab work for more information.

    June 2nd: 3:30-4:30… Getting prepared for our next procedures, and planning a second human practices interview with Dave Bruha, and looking into interviewing other people as well. Did a mini-prep today with our cultures. To look at our procedure, see the lab work for more information.

    June 4th: 3:30-5:00… Worked on some modelling and did our restriction digest. Also completed the interview with Dave Bruha of the Consort Enterprise as a part of our Human Practices portion of our project. To look at our procedure, see the lab work for more information.

    June 5th: 3:30-5:00... We used a spectrophotometer to get an optimal density reading of our newly growth cultures. To look at our results, see the lab work for more information.

    June 6th: 3:30-6:00... At lunch today we did a ligation, followed by the making of competent cells and a transformation of the plasmids into the cells after school. We're letting them sit over the weekend, and then see if they glow red on Monday. To look at our procedure for our ligation followed by the competent cell transformation right after, see the lab work for more information.

    June 9th: 3:30-5:30… Our RFP cells didn't glow red, and we determined that something went wrong during the transformation stage - we are going to try it again and see what exactly went wrong.

    June 10th: 3:30-5:30… We finished making the competent cells, then did the ligation, competent cell making, and transformation using the same protocol as on June 6th.

    June 11th: 3:30-6:30… Our cultures still aren't glowing red after trial number two. We contacted Emily Hicks for discussing our problems, and she gave us advice and trouble-shooting tips. We think that it was the competency protocol that went wrong, as well as the level of growth that our cells were at. When we did the optical density test, our results varied from 0.7-1.2 range, and they should be at about 0.4 for the best growing rate. And so after re-growing our E. coli, we did a single optical density test and got much more optimal results, which may be to blame for the lack of RFP transformation.

    June 12th: 3:30-11:00… Got several more protocols in place and reviewed our handbooks for tomorrow, including the making of competent cells and transformation using a new method, and figured out the variations of the concentration of the CaCl and the different types of growth media added to the cells. We then plated them out on different types of media including SOC, SOB, and LB.

    June 15th: 10:00-5:00… After our third trial, our cells still don't have the RFP in them. Today we researched more troubleshooting solutions for both transformations and for making chemically competent cells, and then we prepared to make more competent cells using a revised method and using new values from Emily Hicks, and this was followed by some poster brainstorming and fixing up the wiki as we waited 2 hours for the LB Broth mixture to sit on the shaker table. In the photo, the plate on the left from this round shows that we do have growth, in comparison to a blank plate with no colonies or growth on it.

   June 16th: 3:30-6:30… Our fourth trial didn't work, and we are for sure that there is something wrong with the transformation or ligation stage, as our Chlor resistance we put into the cells didn't stick, because there were colonies growing on the regular LB plates, but not on the plates with the Chlor on them. We phoned Emily Hicks and asked for some more help... her one suggestion was that maybe something was wrong with the DNA. She's going to talk with her colleagues tonight and let us know tomorrow. Another thing that may be wrong is that our centrifuge holds less than what the protocols ask for, so we had to split it up into smaller volumes, which could be a problem. The photo shows all 16 plates we did under a black light, along with the 2 plates on the far right hand side from the last round - one was with LB media and one was with SOC media.

   June 18th: 3:30-5:00… We have decided to leave our lab work alone for now and focus on the poster, presentation, and wiki aspect of the project. Today we conducted an interview with Lacey Ryan of CARA (Chinook Applied Research Association) and from the perspective of an Environmental Conservation and Forage specialist, she offered valuable advice.

   June 19th: 3:30-6:00… We've been working throughout the day and into the evening on the modelling and on the wiki, as wikifreeze is this Friday. We've also been designing our poster, and have ordered t-shirts for the Jamboree. Progress is going well so far, only a few more things to put up.

Lab Work...

The following are the protocols in which we followed for our lab work, along with the book or web handbooks references, the dates when we did the protocol, and how the protocol went overall.

    ~LB Broth (May 19th): Materials: LB broth powder, Glass bottle. Protocol: Weigh out 20g of LB broth powder and mix with 1L of water. Scale up or down as desired. Autoclave (sterilize) the broth at 121°C for 15 minutes. We made 1.5ml of Broth, and we had no problem following the procedure. ("International Genetically Engineered Machines Competition Guidebook for High School Students and Instructors" ¹ , page 96-97). We also followed OpenWetWare procedures for both the SOC media and for SOB media

    ~Agar Plates (May 21st): Materials: LB-agar powder mix, Plates. Protocol: 1. Weigh out 35g of LB-agar powder mix per litre of media desired. (Note: instead of using powder, we made our own LB Broth powder from a formula given to us by our mentors... 5g yeast extract, 10 g tritone, 10g sodium chloride mixed together.) One litre makes 40-50 plates. Ensure that the mixture volume does not exceed half of the volume of the flask/container used, otherwise it will boil over in the autoclave. 2. Dissolve the LB-agar, using water from one of the wall mounted Nanopure filters. Add a stir bar and use a magnetic stirrer to facilitate mixing. 3. Cover the flask with aluminium foil, and secure the foil with autoclave tape. The foil should be somewhat loose (to avoid building pressure in the flask while sterilizing), but not so loose that lots of liquid can escape. 4. Put the flask in a plastic autoclave tray, load into the autoclave, and sterilize using the 20 minute liquid program. 5. Allow the media to cool until it can be handled. 6. Once media is cool, add other desired ingredients. Use the magnetic stirrer to mix, do NOT add a stir bar now, or the media will be contaminated. (If one wasn't added before, you must do without.) 7. Add the antibiotic that will select for your plasmid. The following antibiotic concentrations are used commonly in iGEM: Ampicillin (stock 100mg/ml, final 100μg/ml); Kanamycin (stock 50mg/ml, final 50μg/ml); Chloramphenicol (stock 50 mg/ml, final 10μg/ml) Note: To achieve final concentrations, add 1mL of stock per 1L of culture media, except for chloramphenicol, where 0.6mL stock per 1L of media is added instead. 8. Pour the sterile, antibiotic-containing broth directly from the flask into the sterile Petri plates carefully to avoid trapping air bubbles. 9. Allow the plates to stand right side up overnight, and then store them at 4°C (in fridge). We had no trouble making these either. ("International Genetically Engineered Machines Competition Guidebook for High School Students and Instructors" ^[1] , page 97-98)

    ~Streaking From Agar Stabs and Growing Up Cell Cultures (May 26th): We made 6 plates, 3 Part A and 3 Part B, following this procedure. After our incubation, our cells were cloudy as the procedure said they would be. ("3A Assembly kit and Transformation Efficiency" ^[2] , page 9-11)

    ~Spectrophotometer Test #1 (May 29th): Using our spectrophotometer (GeneQuant), we tested our cultures to see what their optical density was in order to judge whether they were at their optimal growing stage or after their growing was finished. Our results were as follows... Test blank: LB Broth with Amp and Kan read 0.365. Actual testing: Test Tube 1 (Part B): 0.792, Test Tube 1 (Part A): 1.762, Test 2 (Part B): 0.778, Test Tube 2 (Part A): 0.752, Test Tube 3 (Part B): 0.882, Test Tube 3 (Part A): 1.102. Observation: Part A seemed to grow faster. We were surprised at the huge optical density though, and we considered testing it further but decided not to.

    ~Mini Prep #1 (June 2nd): Completed the Mini Prep with minimal complications - all of our team members were here for this step. ("3A Assembly kit and Transformation Efficiency" ^[2] , page 12-14)

    ~Restriction Digest #1 (June 4th): Overall it went well, and we didn't have to make any modifications to the procedure. ("3A Assembly kit and Transformation Efficiency" ^[2] , page 15-16)

    ~Spectrophotometer Test #2 (June 5th): Our results were as follows for our tubes of E. coli cells that are all supposedly the same... Sample 1: 0.948, Sample 2: 0.736, Sample 3: 0.924, Sample 4: 1.208.

    ~Ligation #1/#2 and Transformation #1/#2 (June 6th and then June 10th): This procedure didn't work. ("3A Assembly kit and Transformation Efficiency" ^[2] , Ligation: page 17-18, Transformation: page 19-20. Note the competent cell making on page 21-26)

    ~Spectrophotometer Test #3 (June 11th): Our results were as follows for our tube of E. coli cells. After talking with Emily we discovered our optical density should be between 0.4 and 0.6 in order for the cells to be at their best 'acceptance rate' for the plasmids they are supposed to take it... Sample 1: 0.404.

    ~ Making Competent Cells, Ligation #3/#4, and Transformation #3/#4 (June 15th): These procedures didn't work. For #3, we followed the protocol step by step to make just 2 plates this time. But when that failed, on #4 we followed the procedure and but also used new values from Emily Hicks, and made 16 plates. Our variables for #4 were 0.1 molar and 0.05 molar of CaCl and we used both LB and SOC media for variety, and we made four with each substance to total 16 plates. We also used heat shock at 42 degrees for 90 seconds, and increased the media added to the competent cells from 250 micro-litres to 500 micro-litres. ("International Genetically Engineered Machines Competition Guidebook for High School Students and Instructors" ^[1] , Making Chemically Competent E. coli Cells and Bacterial Transformation page 95-96)

Workshops...

    This year we have had three workshops with mentors from Calgary. The three of these workshops combined have been invaluable. Between meeting in person and the dozens of emails that have been sent back and forth, our mentors have been priceless to our project and our own personal learning. Despite the fact that our learning curve is nowhere near a plateau, we have come so far and are extremely grateful for the expertise and guidance of these amazing people.

   The first was on February 23rd when David Lloyd and Himika Dastidar drove down to Consort in order to get us started.          

We began the morning bright and early by learning about the basics of synthetic biology and iGEM, as many of our team members are new this year. After going through some basic vocabulary, procedures, and concepts, we moved on to topics more specific to our project. We spent the afternoon looking up parts in the Registry and creating a construct for our Biobrick. After we had the basics, one of our instructors suggested we synthesize a majority of our Biobrick as it would be easier and faster. After determining precisely what would be synthesized, David then proposed that we center our project on a comparison of different reporters and outputs. Enthusiastically, we researched the best choices for reporters and came up with three choices - GFP, AmilCP and LacZ. For the remaining time we had with the expertise of our mentors, we created a skeleton of the code for synthesizing our DNA.

    The second workshop, held on April 5th, was when Emily Hicks and Robert Mayall came down to answer questions and get us started on modelling, more in depth wet lab procedures, and how to improve other aspects of our project.

    We learned about differences and similarities between simulations and mathematical modelling, and the potential programs we could use for them. In order to create a model, we have to gather your 'species' (the possible chemicals or states in your system), create a flow chart for your species where you define how your species interact with each other, and then add general equations to your framework. As for equations, the simplest equation is “A * k = B”, where species A becomes species B at a rate of k. And after gathering the correct information and basing any assumptions that you need to, the next step is to convert your equations to code. From there, you just test and improve, and even compare your model of how you predict the species will react to the real life interactions to see how close you were.

    The third workshop was in Calgary on May 31st and was focused mainly on the presentation factor of our project. Four other Alberta teams - Lethbridge, Canmore, Calgary, and Cochrane - were present as well. The morning began by simply presenting to the other teams, a panel of mentors, and several volunteers. After each presentation the judges and audience would have a chance to ask questions and give feedback on both the content of the presentation and the manner it was presented. It was incredibly neat to see how the other teams were doing and to improve upon our own presentation by listening to the judges comments and the other teams. By the time we had finished all the presentations we broke for lunch and chatted adamantly about what we liked and disliked about our presentation while preparing for the afternoon. After lunch, we were able to meet with the judges individually. Each of them were able to give us tips, suggestions, and comments in much more detail than had been permitted earlier. Finally, we had a session on our presentation skills, with general tips about how to speak to an audience, eliminate nerves, minimize long paragraphs of text, and how to answer questions. For the rest of the workshop we revised our presentations and played a couple rounds of `PowerPoint Karaoke`- an exercise to practice thinking on your feet and talking to an audience.

    References:

1. International Genetically Engineered Machines Competition Guidebook for High School Students and Instructors, 
2. iGEM High School Handbook