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CAROL
Checking Colony Growth on plates
- No single colonies were found on LB+Kan plates. The transformation was done by using XL Gold Ultracompetent cells and plasmid, pCS26+promoters (NEB Quick Ligase).
- Transformation of pBluescript into both XL Gold and Top 10 cells were successful. Cells grew on LB plates.
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EMILY
Ethics Meeting with Dr. Wolbring
- Read two papers on Ethics: First a review paper by Keasling called Synthetic Biology for Synthetic Chemistry, which I didn't find super useful as it mostly just focused on defining Synthetic Biology and metabolic engineering. I also started to read Scenarios for the Future of Synthetic Biology by Stephen Aldrich, James Newcomb, and Robert Carlson. I didn't get too far in this, but it seemed slightly more useful, discussing who will play a role in shaping the field of Synthetic Biology.
- At 10:00 a.m. Mandy, Fahd, Stefan and I met with Dr. Wolbring to discuss ethics. It was an interesting meeting and he has given us a better idea of where to go from here. We discussed the write-up that we did for AIF and he agreed to send us a copy of it with his comments sometime today. He also showed us some interesting tools online that might help us. One of these was a program called uStream where you can record and broadcast videos and invite people to watch and comment. He suggested that this could be a powerful tool to discuss ethics with other teams and guest speakers. We really liked this idea as an alternative to our conference idea.
Sequencing of J13002-LuxOD47E-B0015
- Today I sent one of my colonies down for sequencing. I sent down J13002-LuxOD47E-B0015 colony 6 with the BBk Reverse Sequencing primer. I'm hoping that this result will come back tomorrow and the presence of the B0015 terminator will be verified in my construct. If this is the case, then my circuit will be complete!
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FAHD
Marketing and Ethics for July 23rd 2009
Today, I concentrated my energies at marketing our iGEM project and looking at the ethical aspect of our project. Here is what I did today:
1) I talked to ALPAC. I got her excited about our project this year and told her about that we are developing virtual educational tools. She asked for our sponsorship package so i e-mailed her one. I will call her after August 3rd(ON HOLIDAYS).
2) I had a meeting with Dr. Wolbring till 12.00 pm. I will tell in detail tomorrow what we had discussed in that meeting but here a couple of brief pointers:
>Considering the Economics of Our Project
>Synthetic Biology VS End Product VS iGEM Competition
>Feasibillty of our project
>Degradation of Bio-films VS Degradation of Nano-silver
>Log-term future of energy industry, biofuels
>Patenting: Open Source VS Closed Source
>Ethcis before Benchwork?
>15 Global Challenges that the world faces.
3) I e-mailed Teck Coal Ltd (VP of Engineering and Development). I also did research on the company so I could make specific connections with our project and their organization. I will call heim next week.
4) I found a couple of contacts at Teck Coal Ltd. These include their community relations officer and the Coporate Governance Officer. I will call them on Monday if we our meting ends after 12.00pm
5) I did research on Enbridge Pipelines Inc. I e-mailed a sponsorship proposal to at Enbridge Pipelines Inc. I will get back to him next week
6) I did research on Finning Canada. I e-mailed them a sponsorship proposal at finning Canada. I will get back to him next week.
7) I did research on Focus Corporation. I e-mailed a sponsorship proposal to Focus Corporation and I will get back to them next week.
8) Attended Thane's group meeting.
9) I did research on how to make our ethics online show on ustream TV. I also made an account on uStream TV. I will discuss about the details of this show tomorrow in class.
10) I called VWR if he had received my list of required(Donation) lab reagents and chemicals. He will be out of office till 24th so i will call him next week.
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JAMIE
Testing Competent Cells
pBluescript was used to test transformation efficiency with different amounts of DNA. Amounts were 1000ng, 100ng and 10ng.
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JEREMY
Restriction Digest of PQ-B-R-OU-B in AC to verify its presence
Purpose: to use RD to verify the presence of the PQ-B-R-OU-B construct in the psB1AC3 plasmid. A restriction digest was set up by cutting plasmids from colony 6 and 7 with XbaI and PstI for 2 hours at 37ºC. The digested products were then run on a 0.7% agarose gel (results not shown). No bands were seen on the gel, except for the DNA ladder, indicating that DNA was lost somewhere along the procedure. We believe that after the past few days, it is necessary to restart the construction of PQ and B-R-OU-B into each other.
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KATIE
Still in the Lab
A variable has been added to bacterial transformation so that once the quiz is complete, the object is aware of what DNA was taken up by the competent cells and it opens up the possibilities of using other types of DNA for the activity very easily.
I traced through the entire notecard reader script and I now believe I have a firm grasp about what is happening as the program runs. The dataserver event basically is triggered when there is data being handled at different times, so it will check that the data being handled is a line of the notecard and if it is it will add the string of data to a temporary variable. Then it keeps track of the number of lines read in order to request the next line of the notecard, which requires the number. So now I am confident with the use of this script, which lessens my guilt for implementing it.
I was able to edit and add to notecards that Mandy will read when she has time, which include:
- What is DNA?
- What is RNA?
- Translation
- Restriction Digest
- Ligation
I have also started a display for DNA replication for the base of the spiral when I take some time away from working on the virtual lab, which involves animating a lot of objects to represent the enzymes involved as well as all the pieces that will have to be rezzed to make a complementary strand of DNA. The difficulties I will encounter will probably be with having all objects moving at the right times and where they will have to travel.
I have discovered that my restriction digest activity does more that it has to, which is not a bad thing. So people can just play around if they want to ligate promoter and rbs together for practice or it could be walked through in the instructions. The notecard reader has been implemented in the third and final construction site and tomorrow after group meeting I would like to obtain the names of all the materials required so I can finish renaming the general materials for the activity.
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KEVIN
1. Modelling
Have reviewed all the characteristics that were discussed last time and today wrote a detailed overview of why/how we would implement it, with the rest of the modeling team.
2. Yesterday's Sequencing results
Yesterday, I have sent Pqrr4+B0034 (RBS) for sequencing with BBK CP F primers in order to verify the presence of Pqrr4+B0034. This construct will be used to construct our reporter circuit when the part with GFP+LVA tag arrives.
Legend
Vector contamination
BBK Restriction sites
Sequence of interest
Scar
Sequence of Pqrr4+B0034 (RBS) C1
GGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTG
GAATTCGCGGCCGCTTCTAGAGTATCAGCAAAAACACTACGGTGGATAATCAGTAAAACCATGAAACTAGAGCCCCGCACACTTGC
GGGGCTTTTTAATTTTGAATTTCTTTCTTATTAAAACGCCATTTTTCTGATAAATGTATTAGTAGCAATGCGCATGGTGGCATATT
TGCATCATTTTGCATTTTGCAAATGCGATTTGCAAAATGCGTGCTCAATAAAGCACCAATATGCATCAGGATCGAAGAAAAAAGGC
GTTTTTAAAAGTTGGCACGCATCGTGCTTTATACAGATACTAGAGAAAGAGGAGAAATACTAGTAGCGGCCGCTGCAG
TCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGC
TCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCA
GGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCA
CAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGG
CGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA
AGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACC
CCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAGACACGACTTATCGCCACTG
Sequence of Pqrr4+B0034 (RBS) C9
GGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTG
GAATTCGCGGCCGCTTCTAGAGTATCAGCAAAAACACTACGGTGGATAATCAGTAAAACCATGAAACTAGAGCCCCGCACACTTGC
GGGGCTTTTTAATTTTGAATTTCTTTCTTATTAAAACGCCATTTTTCTGATAAATGTATTAGTAGCAATGCGCATGGTGGCATATT
TGCATCATTTTGCATTTTGCAAATGCGATTTGCAAAATGCGTGCTCAATAAAGCACCAATATGCATCAGGATCGAAGAAAAAAGGC
GTTTTTAAAAGTTGGCACGCATCGTGCTTTATACAGATACTAGAGAAAGAGGAGAAATACTAGTAGCGGCCGCTGCAG
TCCGGCAAAAAAGGGCAAGGTGTCACCACCCTGCCCTTTTTCTTTAAAACCGAAAAGATTACTTCGCGTTATGCAGGCTTCCTCGC
TCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCA
GGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCA
CAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGG
CGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA
AGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACC
CCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAGAC
For both Colony 1 and 9 Pqrr4+B0034 coding sequences, I have verified that Pqrr4 match 100% with the sequence provided by Thane (given when I sequenced last time), and that B0034 also match with the sequence given by the partsregistry.org. We can clearly see that scar has been successfully formed between Pqrr4 and B0034. I now have to wait for the part with GFP+LVA tag to arrive to finish constructing our reporter circuit.
Now that I have verified the presence of Pqrr4, I can now sequence Pqrr4+I13500 (RBS+GFP) in order verify and make those cells competent again so that we can test Emily and Vicky’s mutants.
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MANDY
Ethics Meeting
I do, however, remember the ethics meeting as mentioned above which is quite useful. Among many topics we discussed novel ways to explore human practices in iGEM, and gained potential contacts to interview regarding their views on synthetic biology (for example the issues arising from open source biology).
Getting Advice in Second Life
It works on my laptop again after re-installation, this is an awesome accomplishment.
Today, Stefan was able to meet up with Max Chatnoir, the owner of Genome Island, for a couple of questions. We asked her about how she gets underaged students on her island and she said she can only show them demos on the projector, or her almost-18 year old students get accounts on Second Life anyways. We want to find more information about restricting our island as safe and allow teen grid access, which is something we should look into.
Stefan was able to send our wiki and a few pictures to Max over email, and she liked what she saw . She is looking forward to us allowing second life educators to tour our island, which we are tentatively scheduling late August or September. She also mentioned that it would be a good idea for us to relocate our island to SciLands, a cluster of science related islands in Second Life, such that we’d have neighbours with ‘related topics’, allowing for users to reach our island easily from other islands based on their interest in science. This would involve a relocation fee, but it might be a good idea to consider.
I talked with Katie…
So basically, lab missions will never encompass what you’d see as an entire lab procedure (using all the equipment), as it would be difficult to focus on specific parts. We have allowed each piece of lab equipment to be used with ‘general’ ingredients, and these can be used in conjunction with other equipment as the individual sees fit.
Of course, there are also the lab missions which will practice the following:
1. bacterial transformation and maybe cell culture (depends on the time we have)
2. PCR, gel electrophoresis, and sequencing (sequencing is really just an explanation of what it is)
3. DNA extraction, PCR to biobrick, restriction digest/ ligation (building a circuit)
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PATRICK
Lost Proteins
Retrospective Notebook: This entry was not written on this day, but derived later from working notes I made that day.
I filed a support request with Linden Labs (the company behind Second Life), but with some sixty thousand users online every day I didn't imagine that the TetR object that got lost yesterday would be a high priority. This was a decently large setback.
Not wanting to wait around for my work to rematerialize, I wound up rewriting the changes I had made since the last time I had saved. I also got to work on a new version of the stayput script... with a new provision to stop objects from floating away more than a short distance! Lesson learned.
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PRIMA
Descriptive Title of What You're Doing
Today:
Novus Biologicals and TransAlta emailed me back and said they’re unable to sponsor iGEM. Novus said that they’re a small company and they have donated the amount that they set aside earlier. I called TransAlta and they said they like our project but due to the economic crisis, they had to cut down their budget; they’re only funding on-going projects within their own companies.
I sent a thank you letters and asked for contact from 10 companies who were unable to help us.
I also sent Noreen Schultz (Ab Community Relations Coordinator) from Company 1a breakdown of our budget and a project description (just as she had asked) after she didn’t quite understand the science behind our project. I showed Thane the description and budget breakdown before sending it. I emphasized that we’re not looking for something big, any donation will be greatly appreciated by the team. I’ll follow up with her on Monday, July 27th.
I also followed up with the following:
Company 1
Emailed Noreen S. She understands our project and currently looking over pkg. Follow up on July 27
Company 2
President is looking over pkg, needs more time. Follow up July 27
BioAlberta
The President was out of the country for about a month; he will get back tomorrow. His assistant said they’ll send the cheque of $1000.00 after he gets back.
Company 3
Called HR but couldn’t get a hold of her, left a msg. Follow up July 24
Company 4
called HR and left a msg, Follow up tomorrow to arrange a one on one meeting
GSK
I called her back and she said that since our project for the competition deals with biofilms and they’re a pharmaceutical company; our doesn’t apply to them right now. I said that it can be used for medicine too but she said since I’m trying to raise money for the competition and the project in that case is biofilm, it doesn’t fit with their goals. But she told me send the pkg anyways so I’ll mail to her tomorrow.
Company 5
Called the Community sponsorship lady and she wasn’t in the office. I left a msg. follow up tomorrow.
Company 6
Called Nancy Laviorette (not sure about her position)….left a voicemail…follow up tomorrow. They’re looking over our pkg.
I also updated the gmail excel document with all the list of companies that Fahd and I have contacted. Jeremy said he’ll send me the list of companies and their contact info soon so I’ll put his up too. Jamie’s supposed to do his soon.
I spent some time with Mandy and Patrick re-working/rewriting the second life section of the newsletter, re-wrote the Lethbridge trip section, edited some pics, etc.
Phone calls took a long time since I called some of these companies every 2 hrs (but don’t worry I didn’t leave a msg every time).
Since Jon’s dad is donating to the team, I gave him our account number for BMO and our address for the BhSc office depending on which method he uses to give us the money. I also wrote up a thank you letter which I need to pass by the rest of the marketing team tomorrow before we give it to Jon/his dad.
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STEFAN
Ethics with Dr. Wolbring
Today was spent having some ethics discussion with Dr. Wolbring. We
talked about synthetic biology being different for Kiesling, Ventor
and Endy. For example, Ventor wants to build an organism from the
bottom up, while Kiesling is more interested in metabolic engineering.
Dr. Wolbring suggested we should keep in mind the economic viability
of our project. What is the reason we are doing this project? Is there
something that is more effective and cheaper than our system for
destroying and preventing biofilm formation? He said the teams don't
really take these things into account and some other things about iGEM
that I didn't really agree with because I feel iGEM is not meant for
useful applications and a product that will sell. We're
undergraduates, I don't think groundbreaking discoveries should be
expected. Anyway, he did offer some help in suggesting some ways we
can go beyond the paper (see Fahd's email).
For Second Life, I fixed up the eukaryotic cell today. I then met with
Max Chatnoir, creator of genome island in order to show her my
endoplasmic reticulum (the Second Life one not one in my body). I did
this because I feel it is important to keep my contacts updated and
also she was having trouble creating one herself. She was super
impressed and I also sent pictures of the lab and synthetic kingdom.
She is excited for the unveiling of our island. Then, she mentioned
that we should join SciLands, a collection of science based islands
that are grouped together in Second Life and Mandy took a look at the
application and we thought this would be an excellent idea.
Tomorrow will consist of the ethics (or lab?) meeting and also the
super secret announcement for Sonja. After that, I will most likely be
continuing my work in the Synthetic Kingdom by brainstorming some more
ideas for engineered cells.
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VICKI
Modelling report and plan of attack
We touched on that briefly today but had to put that on hold to discuss lab work. We’re planning to put together something that includes a description of what we’re planning to look at (Hill equation, static/dynamic behaviour, response time, population mean and range), why it’s useful and how we plan to collect the data to develop the model or quantify that aspect of characterisation.
Making the introduction less superficial
We all know that I’m making a lot of vague claims in the introduction (ie, why coordinated behaviour could be useful in degrading biofilms). These need to be substantiated by something specific and credible. I will address that as much as I can within the next few hours. Other areas of concern include a lacklustre description and comparison of the AHL and AI-2 signalling pathways that needs to be improved (ie, why do we want AI-2 beyond my favourite example of enabling a quorum-based logic gate? I need to mention specific cases where the AHL system does not function while AI-2 does, which can be found in the literature); poor formatting of names that need to be italicised or not (genes vs proteins); a more rigorous description of how and why bacteria use quorum sensing in nature; a better introduction to LuxOD47A, where it came from, how it was developed and in what specific experiment it was used in. This list is by no means exhaustive and I’m sure that more will come up once I address the issues that I’ve mentioned.
Materials and methods and results writing
We discussed this in our paper meeting today. I was still somewhat in thesis mode and I wasn’t sure how much detail to include because I thought that we were just writing this for the wiki. As mentioned in the meeting today, I’ll make it less cumbersome, assume some prior knowledge of technologies such as PCR, and use the descriptions that I wrote for the typed edition of my notebook.
I’m still a little uncertain about the results too – I haven’t focussed on it that much yet because I’ve been caught up in the previous sections. From what I understand, since I don’t have any functional validations (if my cells glowed, I would worry), the sequencing results really are the verification that something happened. I’ll also include my very last gel, which includes the DNA ladder, a negative control, LuxOD47A BBk, LuxOD47A BBk + B0015, and J13002 + Lux… + B0015. This displays everything that we’re contributing to the registry; has a good negative control; and distinct size differences that help validate the results. I’ll have a better sense of whether to include more detail as the section takes form – we had talked about including a gel for every stage of the process, but if I can embody all 3 Registry contributions in one, it would be the least cumbersome to just use that.
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