Team:UIUC-Illinois/Notebook/MeetingNotes

From 2012.igem.org

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<li><a name="meet8" >3/25/12</a></li>
<li><a name="meet8" >3/25/12</a></li>
<li><a name="meet9" >3/27/12</a></li>
<li><a name="meet9" >3/27/12</a></li>
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<li><a name="meet10" >4/1/12</a></li>
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<div id="meetingselection2" class="meetingselection">
<div id="meetingselection2" class="meetingselection">
<center><h2>May-June</h2></center>
<center><h2>May-June</h2></center>
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<li><a name="meet10" >4/1/12</a></li>
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<li><a name="meet11" >3/3/12</a></li>
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<li><a name="meet11" >4/3/12</a></li>
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<li><a name="meet12" >3/6/12</a></li>
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<li><a name="meet12" >4/4/12</a></li>
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<li><a name="meet13" >3/7/12</a></li>
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<li><a name="meet13" >4/8/12</a></li>
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<li><a name="meet14" >3/11/12</a></li>
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<li><a name="meet14" >4/14/12</a></li>
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<li><a name="meet15" >3/25/12</a></li>
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<li><a name="meet15" >4/15/12</a></li>
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<li><a name="meet16" >3/27/12</a></li>
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<li><a name="meet16" >4/17/12</a></li>
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<li><a name="meet17" >4/22/12</a></li>
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<li><a name="meet18" >4/24/12</a></li>
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<div id="meet11" style="display:none">
<div id="meet11" style="display:none">
<textarea rows="30" wrap="soft">
<textarea rows="30" wrap="soft">
-
 
+
iGEM Advisor’s meeting
 +
4/3/12
 +
Proposal presentations:
 +
Uros and Adi: “Biodegradation of Polyester Polyurethane by Endophytic Fungi”
 +
- Isolate the genes that degrade plastic and put them together in E. coli to
 +
breakdown plastic at a commercially viable rate
 +
- Alternatively, certain strains of fungi have the ability to degrade polyurethane
 +
plastic.
 +
- We would focus on one plastic composite to degrade. That would make the
 +
project more feasible for one summer.
 +
- E. coli might not be able to handle all of this. It’s been proven that the process
 +
works in anaerobic and aerobic conditions, so yeast might be another object.
 +
- We have the capability to build the type of filtration system necessary.
 +
- Is it the best to degrade this plastic? It’s carbon sequestering. The
 +
degradation of plastic would need to be coupled to something that would get
 +
rid of the CO2. We could couple it to make a precursor for some new plastic.
 +
We would need to repolymerize it in a type of biological recycling.
 +
- Plastic is good to recycle. Another way to think about this project would be to
 +
microbially recycle plastic.
 +
Divya: “Transplastomic antibacterials”
 +
- Plants make small molecule compounds that act as antimicrobials against
 +
Gram positive bacteria. There compounds typically act in combination.
 +
- Genetically modified plants would produce antimicrobials. New genes are
 +
inserted into the chloroplast DNA.
 +
- Use a gene gun to deliver the DNA to the cells. This way the chloroplast’s
 +
double membrane is penetrated.
 +
- Rate of transformation of plants is very low and growing plant tissue is difficult
 +
and takes a long time.
 +
- Transforming plants is a task in and of itself. We do have resources on
 +
campus to do this. It would look very impressive if it actually worked.
 +
- Great project, but we need some feedback from alternative sources about the
 +
gene gun and plant transformation.
 +
- This is a complex project for the time frame. You have to isolate the
 +
compound, look at the genes, then isolate and transform. This might work as
 +
a side project though, if a very simple compound was used and we changed
 +
its expression levels.
 +
Cara: “A Privy Understanding of E. Coli”
 +
- Fecal matter is the main source of bacterial pathogens in waste. Create a little
 +
package of genetically modified bacteria that would be added to a bucket that
 +
would be used as a toilet. Bacteria would sanitize the fecal matter and a color
 +
change would indicate full breakdown.
 +
- How to focus the project: Choose one compound to degrade, look at what
 +
types of bacteria/fungi/protozoans are in the mix.
 +
- Animal feces might be a bigger problem than human waste.
 +
- The bacteria sanitizing the fecal matter would have to compete against the
 +
pathogenic bacteria already in the environment and fecal matter.
 +
- Look up basillophila.
 +
Announcements:
 +
- In the next 2 advisor’s meetings, decide which projects are the best ideas,
 +
focus on those, and represent them in groups. Have different people both
 +
critique and endorse the different projects.
 +
o Rank the projects.
 +
- There will be one main project, and then one good side project.
 +
o The side project functions as both a safety in case the primary project
 +
fails, and as a continued project for the next year.
 +
- Keep in mind what looks good in competition. The work we’ve put in might be
 +
enough, even if there is no finished final project. But the research and
 +
groundwork must be excellent in that scenario.
 +
- Final advisor’s meeting is Tuesday, May 1. At this meeting the main and side
 +
projects will be finalized.
 +
- Next Tuesday we are meeting Dr. Robinson.
 +
- April 17th, we will have a group picture for the brochure. Dress up!
 +
- On Sunday’s meeting we will vote on what projects we will focus on pitching
 +
again. This is the opportune time to add more to your presentation. We’re not
 +
picking 2 projects, we’re picking several to re-research and reevaluate.
 +
Remember, we’re objective and no project is a bad project!
 +
- This Sunday Angela will also explain about the standard cloning procedure.
 +
- Sunday meeting will be at 8PM in the BIF.
</textarea>
</textarea>
</div>
</div>
<div id="meet12" style="display:none">
<div id="meet12" style="display:none">
<textarea rows="30" wrap="soft">
<textarea rows="30" wrap="soft">
-
 
+
iGEM Engineering Council
 +
Elections
 +
4/4/12
 +
- We removed 10 inactive groups (they had not communicated with EC for a full
 +
year)
 +
- Amendment passed, says candidates on co-op or study abroad can make a
 +
video communication to run even if not physically present at the EC meeting.
 +
- Gamma Epsilon general engineering honors society appeals to come off of
 +
suspension
 +
o There is drama and arguments from both sides
 +
o Written, secret vote  They are still on suspension.
 +
- Election results:
 +
o Pres: Courtney O’Connor
 +
o VP: Patrick Kennedy
 +
o EXPO: Elena
 +
o EOH Director: Gloria Lin (voted to allow a non-engineering on EC)
 +
o Director of Leadership: Steven Marks
 +
o Secretary/Treasurer: Troy Meeham
 +
o Dean’s Student Advisory’s Committee Director: Akash Shah
 +
o Future enrichment opportunities: Kritika Jetkey
 +
o EIB Director: Douglas Podgorny
 +
o SITE director: Mary Kate Krouse
 +
o Awards director: Samantha Tone
 +
o Publicity Director: Rachel Gross
 +
o Social Director: Rachel Seidner
 +
o Service Director: Sara Moshaga
 +
o Director of Information: Sid S.
 +
o Knights of Saint Patrick: Christopher Massie
</textarea>
</textarea>
</div>
</div>
<div id="meet13" style="display:none">
<div id="meet13" style="display:none">
<textarea rows="30" wrap="soft">
<textarea rows="30" wrap="soft">
-
 
+
iGEM weekly meeting
 +
4/8/12
 +
Announcements:
 +
Isiah, Lab Manager –
 +
The next 2 weeks is about organizing the lab. We need to divvy up the remaining
 +
work. Expect Isiah’s email about the inventory in a few days. Isiah will just finish
 +
cleaning a few more things up and then the lab will be ready to go. 2 books will be
 +
created, one for the glycerol stock and another for primers/buffers/ etc. There will be a
 +
physical copy in lab and one digital one.
 +
Cara, Social, & Anthony, Treasurer –
 +
Thursday at 4 PM is the deadline for applying for SORF. WashU has not
 +
responded to Cara, so we will try to use Uros’ biobrick contact. We will not be able to
 +
take more than 2 trips, and 1 trip would be awesome and enough work. This trip would
 +
be great for human practices and collaboration. Cara will contact Northwestern. U Mich
 +
will ultimately collaborate with us, so that’s our option. We will apply for the money
 +
anyways, because even if we don’t use it the money will just get reallocated back to the
 +
SORF fund. The application will be about how the trip helps our human practices
 +
collaboration.
 +
Cara & Asha –
 +
contact Courtney O ‘Connor about funding deadlines. EC funding works similarly
 +
to SORF, but we need to check deadlines about that. Asha will be emailing Courtney
 +
(outgoing) and Troy (incoming).
 +
Results of the EC elections:
 +
o Pres: Courtney O’Connor
 +
o VP: Patrick Kennedy
 +
o EXPO: Elena
 +
o EOH Director: Gloria Lin (voted to allow a non-engineering on EC)
 +
o Director of Leadership: Steven Marks
 +
o Secretary/Treasurer: Troy Meeham
 +
o Dean’s Student Advisory’s Committee Director: Akash Shah
 +
o Future enrichment opportunities: Kritika Jetkey
 +
o EIB Director: Douglas Podgorny
 +
o SITE director: Mary Kate Krouse
 +
o Awards director: Samantha Tone
 +
o Publicity Director: Rachel Gross
 +
o Social Director: Rachel Seidner
 +
o Service Director: Sara Moshaga
 +
o Director of Information: Sid S.
 +
o Knights of Saint Patrick: Christopher Massie
 +
Adi –
 +
Meeting with Dr. Amos Monday at 8 am. We’re not looking at funding, but using
 +
facilities instead. Website new, Alex has been sent designs from past years and he is
 +
now requesting filler data and specifics on what we want included on the website.
 +
Everyone should write a short bio and find a picture to send to Asha. Asha will compile
 +
these and the meeting minutes to send this to Adi and Alex. Send these by Friday. We
 +
actually did have a website in 2010, so that will be consulted.
 +
For entrepreneurship, we need to find a faculty advisor. Bob emailed Rao’s contact, but
 +
she never followed up. Angela will contact her professor who is interested in bioethics.
 +
We also need to register for the entrepreneurship competition in the same way that we
 +
applied to the normal iGEM competition. Apply without the membership code and
 +
Courtney will approve of us later.
 +
Divya –
 +
practice time for undergrad research symposium will be on Tuesday. Uros,
 +
Angela, and Divya will be presenting. The symposium is on April 11, from 9-3/4. It is an
 +
hour poster presentation. We are now registered for quad day.
 +
Bob – Has created an entirely new page to start the website from scratch.
 +
Angela –
 +
For the next advisor’s meeting we are meeting Dr. Gene Robison in the first floor
 +
of the gatehouse. From 3-5. Dress casually, but not sloppy. At the end of the meeting if
 +
time permits, Courtney will discuss how to keep a standard lab notebook for us.
 +
April 28th is the chemistry outreach demos that Courtney is doing. The demos will be for
 +
high schoolers. Example demos are the vomiting pumpkin (elephants toothpaste) and
 +
the exploding gummibear. We will be going to Rantoul for this, but transportation is
 +
providing. Ideas for demos: color changing solutions, liquid nitrogen/soap/water, make a
 +
pickle battery, fire in a pumpkin. Cara will contact Dr. Ray of Chem 104 for demo ideas
 +
and protocols. Concrete times and a date for practice will be announced at the next
 +
advisor’s meeting.
 +
Entrepreneurship and human practices needs to draft a proposal. These proposals will
 +
get presented to the advisors on April 24th. On April 22nd we will practice these
 +
presentations.
 +
Agenda:
 +
UIUC-Illinois Judging Form
 +
- Bronze: submit a biobrick
 +
- Silver: characterize biobrick
 +
- Gold: Characterize a biobrick that is not for our team.
 +
- Be sure to familiarize yourself with this! We’re going to continually consult this
 +
during the summer. We are going to get a gold medal!
 +
Lab updates
 +
- Bhalerao: Isiah and Anthony prepared electrocompetent cells. The experiment
 +
will begin Monday/Tuesday this week.
 +
- Jin: Last Wednesday Dr. Jin assigned us to post-doc Dr. Lee. Tomorrow Uros will
 +
meet with Dr. Lee to plan how the project will be executed. Tuesday from 9-11
 +
everyone will meet to replicate the experiment.
 +
- Brad: Cara has been working hard every day. She transformed some cells with 3
 +
different plasmids that each have a his tag and then either GFP, FoA, or LacZ.
 +
The LacZ failed and is being redone.
 +
- Rao: Angela is working with 2 operons to see if a deletion will effect expression.
 +
She tried out 4 different cloning methods. If the PUF project is done, one of 2
 +
specific cloning methods needs to be used.
 +
Proposal discussion:
 +
- Next Tuesday is the second proposal round.
 +
- Isiah: Original idea: Use genetically modified E. coli to modify crude oil/biofuel to
 +
refine/recycle oil. Naturally occurring bacteria would be used. Further research
 +
indicates that time issues make this project infeasible over the summer. Current
 +
research being done has revealed that it is really difficult to grow this stuff in the
 +
lab. Also, one of the reagents to get the bacteria to grow is very harmful for
 +
humans.
 +
o Will’s feedback: For anything biodiesel look at University of Washington.
 +
This is a complex proposal, so to make this successful go look at past
 +
successful biodiesel projects and try to improve it/continue it slightly.
 +
- Uros: Magnetic bacteria.
 +
o Will: Can we use magnetic bacteria in a machine? Use b fields to control
 +
the bac or use the bac to signal things through b fields. These are ideas of
 +
applications.
 +
- Adi: Degradation of plastic composites.
 +
o Will: An Australian team has worked on this before.
 +
o Potential for a great human practices project: Write reviews on different
 +
topics (biofuels, sensing, etc) to help future iGEM teams orient themselves
 +
each year. That way it makes it easier to see what work has been done
 +
before. Also, we could devise a strategy to organize and exercise quality
 +
control over the parts registry. Start with a call/teleconference survey and
 +
then recompile.
 +
o Stanford-Brown created a human practices website and an alumni
 +
website. Max Song did this, and we’re going to try to contact him for
 +
further information and collaboration.
 +
- Angela: update: she has all of the PUF protein sequences and the RNA/aa info. It
 +
can be synthesized by a company (time and money) or self-synthesize (2 weeks,
 +
little money). University of North Carolina is researching the PUF protein and so
 +
we could ask for a template DNA PUM1 gene from them. Jin’s lab could also
 +
PCR out the PUM1 gene that expresses the PUF protein. So overall there are 4
 +
different sources to get the gene. Translational repressors are an application.
 +
GFP can be used to characterize. Small side project – design and optimize a
 +
construct for the project (to be presented in the next advisor’s meeting).
 +
- Divya: looking at things with microbial properties, but not plants. Maybe yeast.
 +
- Bob: Found a part that uses light to run an ATP pump. We could look into
 +
photosynthetic bacteria rather than interfacing the electro-animal/slug with
 +
bacteria.
 +
- Uros: There are fungi/bacteria that can degrade plastic via serine hyrdolase. Yale
 +
is probably going to do this project for iGEM. It’s a simple but pretty cool project.
 +
We can anticipate that this will be a popular idea. Collaboration with Yale is an
 +
option.
 +
- Anthony: Looked into the price of the substrate ARA, but is unsure about how
 +
much is needed. Also, there are troubles getting e. coli to take up the fatty acids.
 +
- Asha
 +
o Broad review of how tuberculosis is treated. Look for someone on campus
 +
who is researching on how to treat tuberculosis/bacteria. Look for a
 +
direction to research further.
 +
- Cara:
 +
o An Australian team has put e. coli in sewage water before. It would be a
 +
2010/2009 team. So we can look into improving that project.
 +
What we will be further researching:
 +
PUF project – Angela, Divya, Asha
 +
Plastic degradation – Adi, Uros,
 +
Fatty acid producing e. coli – Anthony, Isiah
 +
E. coli waste management – Cara, Bob
 +
The next time these ideas are presented – genes and a schematic should be presented.
 +
Next weeks:
 +
- April 10 –meeting with Dr. Robinson
 +
- April 15 – general meeting to prepare for the second round of advisor’s
 +
presentations
 +
- April 17 – advisor’s meeting for second round of presentations
 +
- April 22 – general meeting for the third round of presentations
 +
- April 24 – advisor’s meeting for 3rd round, entrepreneurship and human practices
 +
proposals
 +
- April 29 – quick update to everyone and a quick summer plan
 +
- May 1 – last meeting! The advisor’s meeting to give updates and feedback on
 +
our main and side project. Entrepreneurship and human practices will also be
 +
discussed. This is when we will plan our summer schedule and agenda.
</textarea>
</textarea>
</div>
</div>
<div id="meet14" style="display:none">
<div id="meet14" style="display:none">
-
 
+
<textarea rows="30" wrap="soft">
 +
iGEM Saturday meeting
 +
4/14/12
 +
Project Palooza!
 +
Microbial Expression and Characterization of a Sequence-specific RNA-binding Protein
 +
for Translational Regulation
 +
- Express PUF in prokaryotic cells and turn it into a biobrick
 +
- Can we use PUF as a translational repressor? This would allow for a more
 +
rapid change in protein levels.
 +
- Using PUF is a relatively new idea. We would need to study how good the
 +
affinity of PUF proteins are. (Binding works so that 2 amino acids interact with
 +
1 nucleotide. A 16 amino acid PUF protein recognizes 8 nucleotide
 +
sequence.)
 +
- Characterization: Artificial 8 base pair design for inhibition of gene expression.
 +
- Optomization: Make a control, and then one nucleotide sequence with 1 one
 +
bair pair off. Study the affinity of PUF to both sequences.
 +
- Biobricks: 1 biobrick for each of the PUF repeats (8 in total), multiple
 +
recognition sites, modular devise for translational regulation.
 +
- This is a significant project, but need to look into applications. That just makes
 +
it that much cooler. Compile ideas and send to Angela by Monday night.
 +
Resveratrol and Piceatannol Project (Cara’s new project)
 +
- Resveratrol is turned into piceatannol in the body, which then goes and alters
 +
genes expressions when fat cells mature. It interacts with the insulin
 +
receptors on immature fat cells, stopping them from maturing.
 +
- 1) Can build piceatannol from the ground up (that’s hard.) 2) Use E. Coli to
 +
metabolized piceatannol from wine/grapes/etc (but how much is actually
 +
there? Is it actually efficient to do this?) 3) Put resveratrol in a medium, then
 +
use E. coli that would digest it into piceatannol and secrete it.
 +
o #1 and #3 have both been done.
 +
- Going forward: Look at stability/solubility/degradation. Apparently piceatannol
 +
also inhibits something in humans? What part of piceatannol is actually
 +
interacting with the insulin receptor? (To check on this last one, contact the
 +
Theorectal Biophysics group on campus to see if we can get time on their
 +
computers to do some protein docking modeling.) Edit: An email update on
 +
this has been sent out.
 +
- Application idea: Synthesize piceatannol (somehow) so that it will work in the
 +
body and be able to help maintain a healthy level of body fat. Create an E.
 +
Coli system that will allow piceatannol to work in the human body.</textarea>
</div>
</div>
<div id="meet15" style="display:none">
<div id="meet15" style="display:none">
-
 
+
<textarea rows="30" wrap="soft">
 +
iGEM weekly meeting
 +
4/15/12
 +
Announcements
 +
Bob – Making progress to learn wiki coding and editing. Will also try to edit the wiki with
 +
current ideas and team info.
 +
Adi – meeting with Joe Bradley, Angela’s contact, as a potential Entrepreneurship
 +
advisor. Meeting is at Friday at 1:30. Angela will also be attending. Dr. Amos has also
 +
given card access to DCL to Adi, Uros, and Angela. They also received lab tours and
 +
saw the technology we now have access to. Adi is working with Alex to see how quickly
 +
the website can be up and running (ideal – end of the month). Also has some contacts if
 +
we decide that the PUF application should be HIV. UNC Prof. Tracie Hall will be
 +
contacted to see if PUF has uses with HIV. (Contact by Angela)
 +
Uros - Prof. Ha was contacted over PUF research. Emails have been gathered and
 +
professors have been contacted over potential collaborations. All of the labs have been
 +
quite friendly and receptive. The more opinions the better!
 +
Anthony – Checking with Courtney and Melissa that the books are balanced. The
 +
traveling funds we applied for are the 3 days the Friday weekend of July 13-15. (Leave
 +
Friday, get back on Sunday). Busing and driving are being considered as options.
 +
Asha – will be visiting the Engineering Council office to check out the availability of
 +
funds for the July traveling. Also, the U Mich pres will be emailed about human practices
 +
collaborations.
 +
Angela – Next Sunday the President of the University of Michigan iGEM will be coming
 +
to our meeting! Contacted Mat Song, last year’s human practices director, and that
 +
correspondence has been sent to Asha.
 +
Divya – Undergraduate Research Symposium went well! The pictures from that will be
 +
posted to facebook, and a new twitter and flicker will be created for iGEM to broadcast
 +
our work. During the summer, we will also have a blog on the iGEM website,
 +
spearheaded by Divya and Cara. Divya will also be creating a biography of our work,
 +
focusing on our opinions and perceptions of synbio before, during, and after the
 +
summer. Everyone is invited to the IGB Fellows, but previous members will be
 +
presenting the old E. Chiver poster. Everyone is registered though, so go grab a free
 +
lunch and listen to the talk!
 +
Cara – Let’s have a get together before finals! The tentative date is reading day. This
 +
will probably be our last meeting/get together before dispersing for finals and until May
 +
15th.
 +
Agenda
 +
What’s our timeline?
 +
Proposed timeline: Next Tuesday – advisor’s meeting to present our more specific
 +
project proposals. Human practices and entrepreneurship should also prepare
 +
presentations. April 22 – presentations to update and further refine proposals. April 24-
 +
Advisor’s meeting: Human practices and entrepreneurship give formal presentations to
 +
advisors. May 1 – last advisor’s meeting to discuss final plans for the summer and for
 +
projects. This is the last meeting before dispersing! (In general, weekly meetings are
 +
updating and planning the projects. Advisor’s meetings are for presenting and getting
 +
critiques on these project ideas and plans).
 +
More specific plans:
 +
- Phat project: A solid plan will be developed by the last advisor’s meeting. So
 +
this project will lag behind the PUF project slightly. Phat group will physically
 +
meet Monday night at the UGL.
 +
- Human Practices: We will go ahead with the idea of standardizing the search
 +
for biobricks and past iGEM projects.
 +
- Entrepreneurship: Progress does depend on what the final project will be. At
 +
that point the entrepreneurship advisors will helps us make a decision about
 +
what plan of action is best for the entrepreneurship competition. Once the
 +
project is known and the option is chosen, the application will be a relatively
 +
simple execution. Major project should be made during the summer.
 +
- Wiki: Does a separate wiki need to be done for the entrepreneurship
 +
competition? The wiki template will be set up, so that during the summer our
 +
data will just be slotted in. This minimizes the work done during summer.
 +
- PUF: further applications will be looked into. We will google + hangout on
 +
Monday night to discuss the proposal presentation. Take the initiative to
 +
contact UNC researchers if necessary.
 +
Next advisor’s meeting! Dress up to take a formal picture (that means suits and jackets!)
 +
Tentative agenda: Brief discussion of ideas for entrepreneurship and human
 +
practices. Then PUF and Phat present, followed by a great deal of discussion. We will
 +
also ask if it is feasible to do a main project, a side project, and the entrepreneurship
 +
competition.</textarea>
</div>
</div>
<div id="meet16" style="display:none">
<div id="meet16" style="display:none">
-
 
+
<textarea rows="30" wrap="soft">
 +
iGEM Advisor’s Meeting
 +
4/17/12
 +
Project Proposals Round 2
 +
Resveratrol Metabolite Blocks Apidogenesis
 +
- Why aren’t we just producing lots of resveratrol and putting it in the body?
 +
The body will naturally change it into piceatannol for us. Response: Does it
 +
happen in the necessary quantity and concentration? We want to make these
 +
molecules more stable and more soluble in the body.
 +
- Resveratrol is widely regarded as a wonderful compound to extend life and
 +
help health. It’s widely researched and popular, but there is much controversy
 +
about it still.
 +
- How are we going to make this more soluble? Add groups to the non-active
 +
sites of the molecule. Groups increase solubility, like halogenation.
 +
- Do these modifications change the molecule itself? Well we can run
 +
computational tests on that. We can also biologically make resveratrol
 +
derivatives and test them like a pharmaceutical company.
 +
o Beyond looking a solubility, computational testing might not be of much
 +
help. You really need to synthesize libraries of the molecule and test
 +
them.
 +
- Need to look into P450 enzymes. But we want to stay away from protein
 +
engineering. We can buy and test a range of enzymes though.
 +
- Computational side? The molecule docking technology to do simple hydrogen
 +
bond analysis is free, but for the more complex and detailed information we
 +
need an expert. Courtney will consult with one person in her lab who has
 +
experience in this area.
 +
o The computation sounds like it is beyond the scope of the summer.
 +
o Our best bet in terms of time is to just make a few analogs and test
 +
them.
 +
Microbial Expression and
 +
- PUF works with only 8 base pairs of recognition.
 +
- Where should the PUF binding site go? Let’s test a bunch of models with it in
 +
various places to see which is the most effective (via visibility of YFP).
 +
- Need to clearly understand the PUF mechanism. This ensures that it is
 +
binding to a specific RNA sequences and not just the PolyA tail.
 +
- Applications: the PUF library is obvious and useful, but could also use PUF as
 +
a scaffolding tool in the style of zinc fingers.
 +
- How about engineering different kinds of PUF? First need to make sure it
 +
works in prokaryotes, then test the first type of PUF and optimize where the
 +
binding site should be. Then and only then we should look at making different
 +
versions of PUF and starting the library.
 +
- For this to work at all, it is necessary to make really sure that PUF works in E.
 +
coli.
 +
o It is possible to make this work with cDNA. Are we sure that we can get
 +
the cDNA from UNC?
 +
- It is a small protein. It might be easier to just synthesize it rather than clone it.
 +
We should definitely start with cDNA.</textarea>
</div>
</div>
<div id="meet17" style="display:none">
<div id="meet17" style="display:none">
-
 
+
<textarea rows="30" wrap="soft">
 +
iGEM Weekly Meeting Minutes
 +
4/22/12
 +
Announcements
 +
Angela – New advisors! Kori Dunn, Angela’s graduate advisor in Prof. Rao’s lab. Prof.
 +
Joe Bradley of Business and Administration is our new Entrepreneurship advisor.
 +
Everyone needs to sign up for the iGEM dropbox, there are important papers and
 +
documents there that are useful for everyone. We will video chat with Dr. Wong will
 +
happen this Tuesday from 3-4 at the 2nd floor of the gatehouse. If the advisor’s are
 +
available, we will have a normal advisor’s meeting from 4-5 (this is tentative, watch for a
 +
confirmation email). Angela has received the PUF plasmid from UNC and is looking to
 +
see if the transformation is successful. If the transformation is successful we will move
 +
onto designing the construct. University of Michigan iGEM president is coming down
 +
this week. When will we meet with him?
 +
Isiah – not present, but everyone needs to send him the categorized inventory
 +
information!
 +
Adi – Met with Joe Bradley. PUF and Phat projects were discussed, but mainly PUF. If
 +
the PUF project is successful, we will have made steps towards making a toolkit. We
 +
have the option of marketing our toolkit or targeting a specific disease. Our first step is
 +
intellectual property. The patent database, USPTO, and Espacenet will be searched to
 +
see if any patents exist already. If we choose the business plan model, we will market
 +
our team as though we are a company. We will discuss how we will start and grown our
 +
company.
 +
Divya – An email has been sent out about the IGB symposium. Last year’s project will
 +
be presented at the poster part of the day by 2 former iGEM members. If you want free
 +
breakfast and lunch, register! Our theme is “other IGB.” The brochure will be further
 +
discussed with Melissa.
 +
Asha – The final Engineering Council meeting of the year is next Wednesday, and the
 +
retroactive summer travel funding will be discussed. Human practices has contacted
 +
Northwestsern, Brown-Stanford, and University of Michigan to collaborate. Instead of
 +
building a whole new website, Angela will contact Alyssa (Cornell) who is an
 +
administrator of the Alumni iGEM site to see if we can create a new page on their
 +
website. The point is to collaborate, not compete. In the fall we will also look into
 +
potentially volunteering to teach middle schoolers/ high schoolers about synthetic
 +
biology.
 +
Bob – Will be working to put all of the meeting minutes up on the wiki. The website
 +
looks basically the same as last time, but David has been contacted about how to make
 +
the wiki look more like a website through templates. Alex and Bob will share contact
 +
information to make everything look awesome. We also want to put our ideas on the
 +
wiki so that we can have them available for everyone to see the time-stamp of when we
 +
came up with it.
 +
Anthony – Estimated budget to travel to North Carolina for next year’s IBE conference.
 +
Cara – There is concern that the proposed phat project is too roundabout. A better idea
 +
might be to simply mutate the cytochrome enzyme that converts resveratrol to
 +
picetannol by sending it out to a company and then just testing all of the different
 +
versions. Tentatively, it sounds like this is a long term project. It would be better to make
 +
1 of 2 simple constructs to test.
 +
Social stuff: We will have dinner on Reading Day together. Go to one place for actual
 +
food, then after to a dessert place!
 +
Will’s comments – As an RSO we can set up our own poster session about our iGEM
 +
projects and synthetic biology. We could do it at the end of the fall semester and invite
 +
other research groups who would like to present at the end of fall semester (there aren’t
 +
many symposiums at that time.)
 +
Agenda
 +
Bootcamp:
 +
- Are we going to be ready to go into lab? Who is good at what? We need to
 +
start thinking about how we are going to divvy up lab work.
 +
- We need to write out specific protocols.
 +
- We will partner up.
 +
- We will set aside time at the beginning of the summer to simply write out
 +
protocols and seriously and thoroughly learn all of the procedures.
 +
- Overall, we need a good wetlab plan as soon as possible.
 +
PUF stuff :
 +
- The PUF sequences have been put in the iGEM dropbox.
 +
- Why do we need the PUF sequences? Because we need to design primers.
 +
o Yellow = 8 PUF repeats
 +
o We got this from UniProt, searching for PUF1. The information from
 +
this site has been put on the Word File in the dropbox.
 +
o An alternative search site is the NCBI, using search “Protein”.
 +
- Biobrick foundation website has lots of assembly methods. Using RFC, we
 +
get the biobrick prefix and suffix for the PUF coding sequence. These
 +
prefix/suffixes contains restriction sites. *****put website from the green
 +
handout here ********
 +
- Plasmid backbone: pSB1c3 (this is what we submit our biobrick in).
 +
pSB=plasmid synthetic biology. C= colminithol resistance. A map of this
 +
backbone can be found on the parts registry to physically see the restriction
 +
sites.
 +
- Annotation is getting sent to everyone.
 +
</textarea>
</div>
</div>
<div id="meet18" style="display:none">
<div id="meet18" style="display:none">
-
 
+
<textarea rows="30" wrap="soft">
 +
iGEM Advisor’s Meeting
 +
4/24/12
 +
Conference Call with Dr. Zefeng Wang_of UNC___
 +
- Slide #2 – 2 constructs (one with PUF binding, one without). The one without
 +
the PUF binding site gives YFP because it can be translated. Dr. Wang
 +
wonders if this will this work?
 +
- Currently there are no papers about the prokaryotic recognition sequences.
 +
Dr. Wang thinks it will not work. He recommends changing to a reporter that
 +
will work.
 +
- Papers suggest that PUF binds to the 3’ UTR in eukaryotic cells. We are
 +
unsure of if this will work in prokaryotic cells. No one knows exactly how PUF
 +
works in eukaryotes – they recruit other factors that stop translation. These
 +
other factors might not exist in prokaryotes.
 +
- There is a paper that claims PUF has worked in prokaryotic cells, but no
 +
plasmid was included in that paper.
 +
- Yeast 3 hybrid system works well in Dr. Wang’s lab. He can provide this as a
 +
modular system to start with. This saves us the trouble of what if the recruiter
 +
doesn’t work.
 +
- Many PUF mutants can be nicely expressed in E. Coli. Some cannot be
 +
expressed.
 +
- We can also try ASRE – artificial site specific RNA endonuclease. But we
 +
wanted to do it in E. Coli.
 +
- Dr. Wang ran an experiment where PUF was used to repress Bgalactosidase.
 +
The PUF binding occurred in the gene. The lower expression
 +
is from RNA degradation. PUF doesn’t cut RNA, just bind it. Dr. Wang put the
 +
PUF together with an endonuclease that cut the RNA when PUF bound to it.
 +
But that is not PUF anymore, it’s ASRE (the name of the fusion protein).
 +
- The whole ASRE complex looks like: Flag-PUF-linker-endo
 +
- Dr. Wang’s lab has a library of these ASRE’s that they are willing to share with
 +
us. But the paper is unpublished, so the data (if it works) needs to be treated
 +
confidentially. We would not release the information to the iGEM website until
 +
everything is collected and we have conveyed the results to Dr. Wang.
 +
- Complications: We don’t know what the rate-limiting step yet? The binding or
 +
the cut? Change design so that PUF binding site is between the RBS and the
 +
gene. We put the PUF binding site multiple times to try to increase the
 +
effectiveness. Could rearrange so that the reporter is under the repressor.
 +
- Distance between binding and digestions sites is very close.
 +
- To add multiple binding sites, where would they go? Can we add one to the 3’
 +
UTR end so that it’s after the stop codon? Does it work?
 +
- Possible death mechanism: Engineer a PUF between the start codon and the
 +
promoter. It’s a highly conserved region so it will stop the manufacture of
 +
many proteins.
 +
- We can try multiple scenarios. We want to use the existing protein and
 +
change the binding site, as opposed to using mutated PUFs.
 +
- Dr. Wang’s overriding concern is if/how the reporter will work. Is there any
 +
natural repressor in the protein? Could use Lac I and B-galactosidase. Want
 +
to arrange it so that we control gene A, gene A and YFP have inverse
 +
expression. Once this works, changing modules will be easy. We will
 +
investigate more cases like Lac I.
 +
- Dr. Wang already sent the construct. But Dr. Wang will look into sending and
 +
MTA over to Dr. Jin.
 +
- Dr. Wang’s lab has every possible ASRE in a library. This is a good thing to
 +
look into further down the road. Then all we would do is test the readout from
 +
the different ASRE.
 +
- Question about plasmid Erin sent: It is for eukaryotic expression but it does
 +
replicate in E. Coli. It is PUF with another fusion that changes splicing in
 +
eukaryotic cells. But now we need the new plasmid with the fusion protein
 +
ASRE.
 +
- Erin used a step-wise amplification system to generate the library of ASRE.
 +
You can make it one-by-one, but the step-wise PCR created (in theory) all
 +
possible PUF.
 +
- Dr. Wang is open to collaboration and will share needed plasmids with us.
 +
- Antibiotic resistance will be Amp (maybe K? depends on which backbone).
 +
- The current backbone has K resistance. 3 plasmids: reporter gene with YFP
 +
that is controlled by inhibitor protein. Gene 2=codes for inhibitor protein and
 +
has PUF binding site.
 +
- 3 markers should be enough. Although we can combine to get just 2
 +
plasmids.
 +
Discussion
 +
- It is clear that binding is not enough. We need the PUF binding and
 +
endonuclease activity. Dr. Wang’s lab will send us their contruct.
 +
- The concern right now is the location of the binding site. Can we choose one
 +
or can we engineer one?
 +
- There are many types of repressors to choose. We need to research which
 +
would be the best choice. For next week we will remodel the construct and
 +
find different repressors. Also look for a biobrick part.
 +
- It’s a big help that they already have the whole PUF library.
 +
- For the RBS we will use the Biobrick. This is because we want to submit a
 +
standard part, so we should standardize everything. Because we have to
 +
ligate everything, it’s a problematic idea to put the binding site between the
 +
RBS and the gene.
 +
- Other idea: Remove the loop area from the end of the mRNA. The 3’ UTR is
 +
always involved in stability, so if our PUF binds on the loop it would be more
 +
susceptible to the nuclease.
 +
Cara’s phat project
 +
- Buy resveratrol and a cytochrome P450 (several have been found, a human
 +
one and a mutant Bacillus one. However the Bacillus one may not be
 +
available for purchase online.) Sigma Aldrich is our potential vendor.
 +
- Resveratrol ranges in price and available styles (approx $100 for 100 mg,
 +
approx $300 for 500 mg)
 +
- The goal is to create mutant piceatannols. So we mutate the cytochrome
 +
P450 to see if we can get mutant piceatannols.
 +
- It’s hard to work with cytochrome P450 in the prokaryotic system because it’s
 +
made to work in eukaryotes. That’s why we can the bacillus cytochrome.
 +
- Three available ways to make mutant enzymes: site specific mutagenesis by
 +
a company, arichrome PCR, chemical mutagenesis
 +
- Overall idea for a lab plan: We put the mutated cytochrome in the e. coli and
 +
feed it resveratrol. We get a chip that binds to the insulin receptor and see
 +
what has binded to our chip to see what is happening.
 +
- Is this too ambitious? Or is it totally possible? There are conflicting opinions.
 +
- Biggest problem is the low solubility. So all of the assays and screenings are
 +
not easy.
 +
- Our ideal mutant would be more soluble, because then it would avoid
 +
degradation is the bloodstream. This random mutagenesis is a bit of a game
 +
of luck. How many mutations do we need to go through before we find
 +
something good? How many residues are there?
 +
- If we have directed group addition or the making of derivatives, it might be
 +
better and faster and potentially yield better results for our mutated
 +
piceatannols.
 +
- Feedback inhibition occurs when resveratrol and piceatannol inhibit the
 +
cytochrome that creates the mutants.
 +
- The painful part is where we need to evaluate each clone by an individual
 +
assay. Is there a way to create a florescence detection method? This is much
 +
easier. That way if the chemical is modified we see a color. Then we can do a
 +
ton of wells and only examine the wells that have changed color.
 +
- What is our desired endpoint? We don’t know what our final product A is.
 +
We’re just looking for the best product, so how can we know for sure?
 +
Extensive testing is needed. There are 2 unknowns (the enzyme and the
 +
compound.)
 +
- This had morphed into a pharmaceutical project, which is time consuming.
 +
- Still a good side project if we can obtain and test various derivatives.
 +
- Can we get constructs from people who have published? Then we can make
 +
sure that it will actually work as expected in E. coli.
 +
- 2 important things: We made a novel chemical and we made it in a
 +
commercially viable amount (g/L). We need to engineer the cell to produce it
 +
in proper amounts. So look at making P450 more stable so it can produce
 +
more.
 +
- Purdue paper: They did their research in cell cultures, but mentioned
 +
degradation in the body. So does it degrade in the cell culture?
 +
Announcements
 +
-New advisors: Kori Dunn (3rd year graduate student from Prof. Rao’s lab), Amet (also a
 +
3rd year grad student from Prof. Rao’s lab), Prof. Joe Bradley for Entrepreneurship.
 +
Agenda for next weekly meeting
 +
- We are wrapping up for summer next week! It is important that we make the
 +
most of our meeting time, so come on time and come prepared!
 +
- Sunday Meeting agenda:
 +
1) Angela finishes up the biobrick talk
 +
2) Vote on projects. Do we want 2 equal projects? A main project and a
 +
side project? Which one is which?
 +
3) Vote on bootcamp. Do we want a bootcamp? Will we split into pairs?
 +
How long will it last and what lab procedures will we cover?
 +
4) Create a solid wetlab plan. Make one for any and all projects that we
 +
have. Start simply yet thoroughly and let the gold medal guildelines
 +
determine our progress. Make sure everyone is clear on this plan and
 +
understands all of the constitutive components.
 +
5) Vote on vacation time. When will we reconvene for summer? Before
 +
coming back to work, what should everyone have done? (Reading
 +
papers, awareness of lab procedures, just take a brain break).
 +
6) If time permits, general announcements, including an update on
 +
reading day’s social outing.</textarea>
</div>
</div>
<div id="meet19" style="display:none">
<div id="meet19" style="display:none">

Revision as of 21:45, 19 June 2012

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