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CAROL
Wiki Updates
- Updated the wiki pages today, so there is full descriptions for all the parts of our project. As well, started working on the protocols for our wiki today.
- The transformation with pCS26 vector and the sigma 70 promoters ligated by T4 Invitrogen Ligase showed small colonies on plates late in the afternoon, left overnight in the incubator.
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CHINMOYEE
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EMILY
Verifictaion Digest of J13002-LuxOD47E-B0015
- Today I did a minprep of my overnight cultures of J13002-LuxOD47E-B0015. I took the concentration and did a restriction digest with XbaI and PstI. I ran this on a gel this afternoon with uncut plasmids of each colony as a control as well as LuxOD47E BBK (uncut) and J13002-LuxOD47E (uncut) as size controls. See gel photo below.
Lane 1- J13002-LuxOD47E-B0015 Trial 2, C1 cut with XbaI and PstI
Lane 1- C 1, uncut
Lane 3- C2, Cut
Lane 4- C2, Uncut
Lane5- C3, Cut
Lane 6- C3, Uncut
Lane 7-LuxOD47E BBK (Uncut)
Lane 8- J13002-LuxOD47E (Uncut)
Outreach/ Ethics
- Stefan, Fahd and I met to talk about Ethics this morning to make a plan of what needs to be done for the Erhics conference. We're planning the conference for some time in late September/ early October, but we want to start inviting people now. So today we made a list of possible speakers as well as people that we will invite just to come and listen.
- We decided that the first step would be to familiarize ourselves with Second Life. So, this afternoon I made an account on SecondLife and explored a little. I walked through the boards with instructions on them to learn how to talk, move, ect. Mandy then gave me access to our Island, so I got a chance to walk around and look at some of the stuff that our team has been looking on. Tomorrow I'm going to explore an ethics area that Mandy knows about as well as try to find some conferences to sit in on to get a better idea of how they are run. Fahd and I need to get planning our Ethics conference so I think it's a really good idea to become familiar with what we can do in Second Life so that we can decide where we want to have it, what we need to build for it, how we're going to advertise it within Second Life, etc.
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FAHD
Second Life Conference, July Newsletter, Education and Outreach:
Today I sent out July letters to the Following comapnies:
1)Nexen Inc.
2)New England Biolabs
3)Alberta Ingenuity Fund
4)Boheringer Ingleheim Canada
5)Apotex Canada
My Ethics teammates and I today discussed some ways to conduct the Ethics conference on synthetic biology in the online virtual world called Second Life.
I also had a meeting with our education and outreach team. We discussed ways to promote iGEM in high schools and universities.
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IMAN
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JAMIE
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JEREMY
Plasmid Isolation of cl lambda and Colony PCR of LuxPQ-B0015-R0040-LuxOU-B0015 in psB1AC3
Plasmid was isolated from an overnight culture of cl lamba using Sigma GenElute Mini Prep Kit. The concentration and purity of the isolated plasmid was then measured with the NanoDrop spectrophotometer. Restriction digest was performed with XbaI and PstI in React 2 (Invitrogen) buffer for 2 hours at 37ºC. Cut plasmid and uncut plasmid were then run on a 1.0% agarose gel, no bands were seen for either column. The isolated plasmid was then specked again, to verify the initial concentration, which turned out to be accurate. An overnight Restriction digest was then set up as described above; however, 600ng of DNA were used instead of 200ng.
A colony PCR was set up with colonies of the construction LuxPQ-B0015-R0040-LuxOU-B0015 in psB1AC3 as the template. Seven colonies were chosen. This PCR was performed with BBK CP F/R primers (these anneal on the BBK vector just outside the cloning site) and LuxPQ F / LuxOU R primers (these are gene specific primers). Due to the long extension time for this PCR (6min and 20 seconds) it is run overnight and then held at 4ºC upon completion. For PCR conditions, refer to PCR performed on July 27 2009. Restreaks of the seven colonies were made and overnight liquid cultures were made so that plasmid can be isolated tomorrow.
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KATIE
A Second Look through Lab Activities
Today was spent going through the lab activities and modifying the scripts along the way. I revised the whole PCR script so that all of it behaves like the general PCR activity and there is not a problem with locating items and the script will no longer proceed if anything is missing, which I believe happened because of a function modification since it definitely did not do that before. Mandy has been having difficulty with the items we have to add although I have not even when we try the same activity, which leads us to believe that the names of her items and the items we need do not match up so we will be comparing names of objects tomorrow.
I also changed the scripts within:
- Phosphatase treatment
- Restriction digest
- Gel electrophoresis (Mandy made some changes here too)
They now search for items the same way as the new PCR machine so the order items are added does not make any difference. I then had to change the PCR machine’s cleaning methods so that more objects were accounted for as necessary and were not deleted before they could be used. I believe this could have also been a problem for some of the items in the PCR machine since they were possibly being deleted before they could be checked since the machine did not recognize the materials as something that needed to be kept.
Unfortunately, I was unable to make significant headway on the DNA replication animation due to all of my time spent in the lab. I was able to build a primase that rez the RNA primers, but I still need to set the positions the enzymes will have to get to along the single-stranded DNA.
I plan to insert instructions into the bacterial transformation activity tomorrow, which now allows for bacteria to be grown on plates if more than six questions are answered correctly, each plate having their own specific textures. When the plates with growth are touched avatars are given a short note card out of the three I made on Tuesday and each still require a little modification, but now avatars can complete this activity. I still need to add deletion to the activity so that inventory can be cleaned out though, but the rest is operational and Mandy was able to complete it successfully. Providing significant instruction for avatars will be very important within the lab so I will get the other group members to try and follow the instructions I have made tomorrow.
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KEVIN
1. Plasmid Isolation
Plasmid isolation of K082003 (GFP + LVA) and Q04510 (inverter) from TOP10 cells
The purpose of isolating these parts is to construct the reporter circuit with K082003 and response circuit with Q04510. Because the plasmids were relatively pure and high in concentration, I can continue with the construction of the Reporter circuit.
2. Construction of the reporter circuit
a. Restriction digest
There were two ways of constructing this circuit: one being cutting out Pqrr4 + B0034 and inserting it into K082003, and another one is K082003 being the insert and Pqrr4 + B0034 being the vector. However, I forgot that K082003 is in a plasmid that has kanamycin resistance, and just went ahead with putting K082003 into the Pqrr4 + B0034 plasmid. This was done by cutting the sequenced Pqrr4 + B0034 with speI and PstI, and cutting K082003 with XbaI and PstI. Apparently though, the BBK CP primers that were made by Thane are only compatible with pSB1A__ vectors, meaning if I had put Pqrr4 + B0034 into K082003 vector, I would not be able to verify it using the current BBK CP primers.
b. Construction
The insert, being the K082003, was put away into the -20 freezer, and the vector, being the Pqrr4+B0034, was phosphotased. The two were than ligated together, then transformed into TOP10 cells.
3. Verification of K082003
Because I have to make sure the cells that iGEM provided/sent us contain the right parts, I have ran the restriction digested K082003 on a 1% gel. This part is about 756bp long, and the following is the picture of the gel:
As you can see, there is a band slightly above 700bp region in the cut K082003 lane, meaning the piece of interest is likely to be present in the plasmid. In the Uncut lane, multiple bands are visible, some of them being nicked and supercoiled plasmids, resulting in running at different speeds on the gel.
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MANDY
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PATRICK
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PRIMA
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STEFAN
Order in the Kingdom
I managed to set up the exhibits somewhat and develop a semblance of
order. I moved everything to eye level since the person is walking
along the path rather than flying. First is the E.coli
that you "kill" using the chat by saying "colicin". The smelly
bacteria follows, although I forgot to mention this in previous
updates. Not that impressive...I just put a script in so the bacteria
emits a colored smoke periodically (smells like pizza). Next is the
GFP jellyfish/bacteria combo. I realized that the jellyfish from which
the protein is isolated from looks nothing like the abomination I have
created so I built an almost exact replica of Aequorea victoria
and segregated them to an area. Then the Bioremediation
Station (pretty good title, eh?) is next, and there's some unfinished
stairs leading up to it.
why did I do it like this? well we don't want to confuse our visitors
right off the bat. I figured "antibiotics kill bacteria" would be an
easy enough to concept to grasp. Also once they know that different
genes can do different things when expressed in bacteria (smelly), and
they can be extracted from animals (jellyfish) then the bioremediation
(an actual application) becomes easier to understand.
There was also a brief ethics meeting today. We all decided that it's
best if we planned out the Second Life conference as soon as possible.
Fahd and Emily are going to look up some ethics places in SL and
listen to some talks to see how they are organized. We are also
brainstorming about what people should be involved in our conference
as speakers. We are hoping to get people like Andrew from DIY Biology
and other important figures. Audience will also be important and other
than SL residents we are hoping to invite some educators that we met
along the way. Also, now that we're helping TU Delft, I bet at least
one person on their team would be willing to help us.
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VICKI
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