Team:UIUC-Illinois/Notebook/MeetingNotes

From 2012.igem.org

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<center><h2>April-May</h2></center>
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<center><h2>April</h2></center>
<li><a name="meet10" >4/1/12</a></li>
<li><a name="meet10" >4/1/12</a></li>
<li><a name="meet11" >4/3/12</a></li>
<li><a name="meet11" >4/3/12</a></li>
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<center><h2>May-July</h2></center>
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<li><a name="meet17">TBD</a></li>
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<li><a name="meet17">5/25/12</a></li>
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<li><a name="meet18" >TBD</a></li>
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<li><a name="meet18" >5/29/12</a></li>
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<li><a name="meet19" >TBD</a></li>
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<li><a name="meet19" >6/1/12</a></li>
<li><a name="meet20" >TBD</a></li>
<li><a name="meet20" >TBD</a></li>
<li><a name="meet21" >TBD</a></li>
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<div id="meet19" style="display:none">
<div id="meet19" style="display:none">
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<textarea rows="30" wrap="soft">
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iGEM Advisor’s Meeting
 +
5/25/12
 +
PPT Presentation
 +
Biobrick with PUF binding site
 +
- Grow on Xgal to test blue/white colonies rather than quantifying LacZ
 +
- Need a plasmid without Lac I already. Easiest way is to get the strain from the
 +
people who made that biobrick already. A different option is to do a Lac I
 +
knockout.
 +
- Rao: Faster and easier to just do GFP expression.
 +
o A blue/white screen is just a yes or no. It’s not a quantitative assay.
 +
o Can do LacZ and GFP in parallel. That’s a stronger experiment.
 +
Potential Plan
 +
- Assemble biobrick
 +
- Then introduce PUF binding site and mutated PUF binding site. Insert before
 +
RBS of Lac I, after RBS of Lac I,
 +
- Assemble last biobrick of PUF+PIN
 +
- The biobrick that we intend to make is made of smaller biobricks that we will
 +
hopefully process all in parallel.
 +
- Different assembly methods?
 +
- Rao: forget fancy PCR things, including Gibson assembly.
 +
o Need a simple control to test the PUF. GFP is a 2 week control
 +
experiment to test PUF levels.
 +
o The modular Lac I/Z system is worth pursuing, but it should be done in
 +
parallel with the GFP experiment.
 +
- The terminators are small, they are going to be hard to cut out of a gel. How
 +
will we tackle small segment assembly?
 +
o 3A assembly is made so that we don’t have to do gel purification.
 +
Therefore we will be fine on the promoters, RBS, and terminators. The
 +
hard part is the PUF sites
 +
o A possibility is to biobrick PUF by itself so that we can simply order
 +
primers and such for it.
 +
o Other option: ignore the existing biobrick BBa_Q04121 and simply put
 +
the RBS and PUF binding site on the forward primer for the reporter so
 +
it’s all together to begin with. The reverse primer would have both the
 +
terminators.
 +
 Constraint – RBS must be no more than 10 base pairs away
 +
from the start ATG. The PUF binding site will be better suited to
 +
before the RBS
 +
 Also, we can expand the biobrick with the addition of each
 +
primer, by adding aroud 40 bp everytime
 +
Biobricks
 +
- PUF is too small to add on its own. Would need to put PUF with the RBS or
 +
something.
 +
- Ideal biobrick: RBS, PUF, and Lac I reporter all together
 +
- It’s too small to biobrick the PUF binding site
 +
o Really makes a problem with creating a PUF toolkit
 +
Final Plan
 +
- PUF binding site with Lac Z
 +
- Mutated PUF binding site with Lac Z
 +
- PUF binding site with Lac I
 +
- Mutated PUF binding site with Lac I
 +
- PUF binding site with YFP
 +
- Mutated PUF binding site with YFP
 +
- PUF+PIN
 +
- Mutated PUF + PIN
 +
- The RBS and PUF binding sites will be in the promoter for the varying gene.
 +
That simplifies putting that tiny sequence into E. Coli
 +
Remaining questions
 +
- How extensively should things be characterized?
 +
o Worry about it when we get there.
 +
- Should we link PUF to another protein domain on top of the endonuclease?
 +
o Don’t worry about it, we need to basics first.
 +
o We have 3 strains of PUF: Wild type PUF (just PUF), PUF with
 +
endonuclease, PUF with mutated endonuclease
 +
PHAT project
 +
- Korean lab is making stocks of stuff and preparing to ship them early next
 +
week. Will hopefully get them on Wednesday. We will also receive a plasmid
 +
map.
 +
o Need to get them a FedEx account number because normal mail will
 +
take weeks
 +
- First step is to biobrick the wild type BM3 (cytochrome P50), and 2 other
 +
mutants (one with the highest activity level and the other with the highest
 +
longevity)
 +
- Second step, redo their assay to make sure that biobrick formatting doesn’t
 +
affect the enzyme’s activity
 +
o How do we measure piceatannol? We don’t have that capability to run
 +
the mass spec.
 +
o Look into measuring via engineering or some way other than
 +
machinery. Look at reduction reactions. Send an email to the Korean
 +
professor to see if they used any other methods.
 +
- Next step, order powder resveratrol. Mix it in the media and see if piceatannol
 +
would form.
 +
o Contact Matthew Koffas because he published about producing
 +
piceatannol in E. Coli. Tell him that despite patenting issues, we just
 +
want any strain for a proof of concept idea.
 +
- Still looking to play around with the chemical drawing software. It would be a
 +
nice theoretical presentation at iGEM.
 +
Announcements
 +
- Isiah: Will start using a virtual lab notebook that everyone should write when
 +
they use something up. There will be a piece of paper on the bench, but
 +
digital is better.
 +
- Daily Team meeting at 5 in the Union basement.
 +
- Advisor’s meeting every Friday from 3-4 PM.
 +
</textarea>
</div>
</div>
<div id="meet20" style="display:none">
<div id="meet20" style="display:none">
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+
<textarea rows="30" wrap="soft">
 +
iGEM daily meetings
 +
Week of 5/29-6/1
 +
5/29
 +
- No work today because of MMG accident.
 +
- PHAT project: switching to a tyrosine pathway that is cheaper and more
 +
suitable for e. coli. Mark (Octochem) is buying us some resveratrol. If
 +
pursuing a bottom up building model, biobrick resveratrol separately because
 +
there is a distinct demand for it.
 +
o Timeline: End of June, have 2 biobricks of the stuff from Koffas’ lab
 +
(4CL. STS, 4 kumarate ligase) (PUCO is the vector where they are
 +
both already ligated together.) going pkumarate -> resveratrol ->
 +
picetannol
 +
o In vivo assays to confirm Korea’s results and then move on. But are
 +
currently needing to cut down from 9 constructs.
 +
- Melissa has contacted Richard Powers on behalf of iGEM
 +
o He’s at Beckman we should go talk to him
 +
- $ 95 pass to get film stuff from art department for the whole semester. Art 250
 +
and we need a separate mike.
 +
- 2010 uiuc igem team has a good synbio video. Take a look at it.
 +
- Entrepreneurship: still don’t know if one or 2 projects. Come up with a
 +
marketing pitch, a team description, and then an elevator pitch.
 +
- Publicity: Divya will make a brochure by the end of the week
 +
- Journal Club: We will have one! It will probably be 30 minutes a week on a
 +
biweekly basis. It is strictly optional, but there are definite benefits for
 +
attending. The first one will be hosted by Angela.
 +
5/30
 +
- Lab Manager: Our inventory is now on a google doc. Whenever you
 +
completely use something, make a note of it!
 +
- Brochure is in progress.
 +
- Entrepreneurship: Adi is going to start contacting companies about
 +
sponsorships. Is going ahead to go and make 2 projects. Adi will also start the
 +
business plan and elevator pitch with advisor Joe Bradley. He will also skype
 +
with Alex about the website at some point.
 +
- Webmaster: Bob talked to Bhalerao. The server is off and in his office. He will
 +
make accounts for access.
 +
- Publicity: Courtney has sent out a template from last year’s brochure. Divya
 +
will update it for us by the end of the week.
 +
- Angela: Will send us abstracts for editing and critique. This will go in the
 +
brochure.
 +
- PHAT: need to get the actual MTA from Koffas. Rao also wants to talk to
 +
Koffas about that. Prof. Ho will be shipping the constructs on Friday, and we
 +
can anticipate their arrival on Monday. Brad suggests doing TCL (thin layer
 +
chromatography). Quantitatively, it will show a big or littler amount, but not
 +
much else. It takes around 45 minutes. We can look into collaborating with
 +
another lab to purify picetannol if necessary. We can also do LCMS or GCMS.
 +
- Angela: Design the forward and reverse primers for the PUF constructs with
 +
Lac I, YFP, and Lac Z.
 +
- Asha: made LB, made electrocompetent cells, did inoculations for PUF
 +
- Divya: made LB, made electrocompetent cells, started brochure
 +
- Uros: transformed PSB1C3
 +
- Adi: met with Courtney, met with Joe Bradley, inoculated for PUF
 +
- We will work in DCL on the weekends. We need to do PCR so we will email
 +
advisor Ting Lu about getting space in his lab.
 +
5/31
 +
- Divya: working on brochure and blog. iGEM teams are following us and we
 +
are following them on twitter! Redesigning a logo
 +
- Can start thinking about a t-shirt design
 +
- Bob: put a drop down menu on the wiki, changed the project page, can put
 +
the twitter feed on the website under outreach
 +
- Cara: emailed out new primers and would appreciate everyone’s feedback
 +
- PHAT project: got sequences of P10 and wild type
 +
- Angela: can use other cloning methods, 3A assembly is outdated and primer
 +
extension has been recommended, for the testing of PUF binding can use the
 +
native plasmid backbone and not just PSB…, it is recommended that we send
 +
things in for DNA synethesis – iGEM is about the design and not the labor of
 +
cloning again and again (Dr. Jin says that if we can prove it works and cloning
 +
is under $500 then we should send it in for synthesis), all of these backup
 +
plans are very beneficial, new PUF primers are coming in soon (estimated
 +
Monday), we need an application for our project!
 +
o Possible application: PUF can be used for identifying and pinpointing a
 +
gene, we could try to combine the PHAT and PUF projects by using an
 +
mRNA sequence with multiple PUF binding sites. The different PUFs
 +
would be linked to different enzymes creating a biological conveyor
 +
belt for faster picetannol production. And then you can control when
 +
this mRNA is being made.
 +
</textarea>
</div>
</div>
<div id="meet21" style="display:none">
<div id="meet21" style="display:none">
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+
<textarea rows="30" wrap="soft">
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iGEM advisor’s meeting
 +
6/1/12
 +
PUF
 +
- The last powerpoint for the plan was using the biobrick format, backbone,
 +
terminator etc.
 +
- We can use a different plasmid for testing though. So we will clone the
 +
expression construct (PUF) that is already in the PET 4.3 structure into the
 +
BL21 strain. Our testing plasmid will have the RBS-PUF binding site-YFP.
 +
This will be a quicker test because the plasmid already has a promoter and
 +
terminators. The primers for these are already order and will arrive on
 +
Monday. The primer includes the RBS, PUF binding site, and restriction sites.
 +
We are cutting with EcoRI and SpeI.
 +
o PET 4.3 is Amp resistant, the protet is Cm resistant.
 +
o We have to check the compatibility of the plasmids. Check this by
 +
looking at the ORI. If they have the same mechanism that will be an
 +
issue (competition and one will win over the other).
 +
o There are biobricks to make this as well, but our overall goal is for
 +
testing to be as quick and easy as possible.
 +
PHAT
 +
- Working on a gene operon to convert tyrosine all the way to picetannol.
 +
- We’ve found that the 2008 Rice team did tyrosine to resveratrol in yeast. But
 +
not all of their biobricks are characterized, so they may not be functional. But
 +
the 3 enzymes are in the registry.
 +
o Try to get these from the registry and try to put it in e. coli. These
 +
probably won’t be in the kit plate, so will order from the registry.
 +
o PCR the genes and build the operon ourselves. That saves the hassle
 +
of the MTA because Koffas might not approve putting his stuff in a
 +
biobrick.
 +
o Rao will still follow up on the MTA but it might not go through. Putting
 +
things into a public registry kind of defeats the purpose of the MTA in
 +
the first place.
 +
- The correct sequences have been obtained from Dr. Kim. All 3 BM3 genes
 +
are 3 KB (big!). So are 4 genes all going to fit in one plasmid? What options
 +
do we have to deal with this? How big is too big?
 +
o For a standard plasmid 10,000 is the max. After that you have to go to
 +
special plasmids (bac). The thing about bac is that they’re single copy.
 +
o Could we take the ORI from bac and put it in a PSB backbone? No, it’s
 +
not that simple. There are other factors.
 +
- If we assembly the operon, it will still be novel.
 +
- NEW IDEA – BIOLOGICAL ASSEMBLY LINE: we have an mRNA that is
 +
never meant to be translated, it is merely a scaffold for tons of PUFs. It has
 +
various PUF sequences that are all linked to different enzymes that are part
 +
of the tyrosine  picetannol pathway. That way all of the enzymes end up
 +
close together, increasing the efficiency and speed of picetannol production.
 +
o Discussion with Bhalerao: He REALLY wants us to get an application
 +
for PUF. This would be a great concrete application to sell. Because
 +
the RNA enzyme cascade doesn’t just have to be with PHAT, it could
 +
also be with any other pathway. And then we can also control when the
 +
mRNA is expressed.
 +
o Concerns: We need at least 3 different PUF binding sites and PUF
 +
enzymes. And then we need to tether things to PUF. How possible is
 +
this?
 +
o Pan Silver has done this. Check up on it. (Slovenia 2010).
 +
o We need to do a simple proof of concept: Make 2 PUFs. One that
 +
inhibits GFP and one that inhibits YFP. And then after that make the
 +
cascade.
 +
o OR: we could do a simple 2 enzyme cascade. Ethanol is 2 enzymes. Is
 +
there one that’s a color change? We can even use Slovenia’s reporter.
 +
(Go find it).
 +
 Will fusion kill the binding activity of PUF?
 +
o How do we figure out the spacing? It’s already figured out by Slovenia.
 +
o We may want to add an artificial loop so it’s stiffer and the spacing is
 +
easier.
 +
o How difficult is it to tether onto PUF?
 +
 First get the Slovenia system, then address this. Figure out the
 +
enzymes and then go from there.
 +
 Slovenia published a paper with Matthew Delisa (Cornell) to
 +
produce resveratrol in E. coli via a DNA scaffold.
 +
 Also need to read the Pam Silver paper and Harvard paper.
 +
- Should PHAT project keep looking into modeling how to make picetannol
 +
more soluble?
 +
o It would be difficult because it comes down to water molecules, which
 +
aren’t great and are complicated because of the hydrogen bonding
 +
networks.
 +
o The calculations will be difficult.
 +
o Not worth the time investment.
 +
o Everyone should really be looking into the RNA scaffolding.
 +
Characterizing
 +
- Needed for silver medal
 +
- Will be replicating Washington’s winning design (biodiesel) with the help of Dr.
 +
Jin’s lab equipment.
 +
- Look at different temps, different sugar concentrations, etc.
 +
Moving forward
 +
- Still need to prove PUF works
 +
- Need to look into creating the mRNA backbone.
 +
o Needs to be very stable! But since it’s non coding it’s naturally more
 +
unstable.
 +
o Normally stem loops will be sufficient to keep the ends safe.
 +
o The stem loops that will dock the PUFs need to be oriented correctly.
 +
They need to be pointing together, not apart. Can make scaffolds in
 +
parallel and test them, but should start by using folding software. There
 +
is RNA software that can predict hairpin structures, but not loops. And
 +
they only give predictions. Look at mfold. It’s on a web server. It
 +
doesn’t give one structure, it gives several and ranks them by free
 +
energies.
 +
 Also consider introducing PUF binding sites in the middle of the
 +
gene.
 +
o Look into strains without RNA endonucleases.
 +
- Find a 2 step color changing process to use as the cascade
 +
o Could look into pigmented e. coli, but really should look at the Slovenia
 +
project (even though they used zinc fingers instead of PUF and zinc
 +
fingers are much bigger).
 +
- Still work on PHAT, it’s our ‘future’ application.
 +
- If we get all that done, then put the PHAT project on the RNA scaffold.
 +
- Finish characterizing the Washington biofuel project.
 +
Future questions
 +
- Does PUF work in E. coli? It’s expressed but does it bind?
 +
- What 2 enzyme system will we use? Does it work in E. Coli without PUF?
 +
Does it work when tethered to PUF? (Hopefully will be color change).
 +
- Have other iGEM teams done mRNA scaffolding?
 +
How is the old work division changing?
 +
- PUF proof of concept: We can make it simpler, and just do as much as
 +
necessary. Don’t worry about making the whole repression system. Just do
 +
with YFP for yes or no. Angela and Isiah will continue with the proof of
 +
concept.
 +
- Uros, Asha, Anthony, and Adi will begin working on the RNA scaffolding.
 +
- PHAT group: Will take yeast biobricks and put them in E. Coli.
 +
Everyone’s Homework:
 +
- Read up on iGEM 2010 Slovenia
 +
- Pamela Silver Paper about RNA scaffolds. (is from Harvard)
 +
- By next Friday, have constructs done.
 +
THE NEW WORKING PLAN
 +
- Biobrick PUF
 +
o YFP proof of concept. The control for this has the same number of
 +
base pairs between the RBS and the YFP gene.
 +
o Biobrick wild type PUF and PUF with endonuclease
 +
- Find a 2 enzyme system that is color changing.
 +
o Make sure it works in E. coli.
 +
o Make sure it works when it’s attached to PUF.
 +
o Look for the simplest possible system!
 +
- RNA scaffolding
 +
o Design the RNA scaffold.
 +
o Run it on mfold.
 +
o Design regulation mechanisms so that we can see it’s better than
 +
DNA.
 +
- PHAT project
 +
o Clone DM3’s into E. Coli
 +
o Biobrick TAL, 4CL, STS, DM3’s for e. coli
 +
o Make the operon that will work in E. Coli
 +
o If time permits: Bind the resveratrol pathway to PUF to use in the
 +
assembly line.
 +
- Characterize Washington’s Biofuel project.
 +
o Qualifies us for silver.
 +
</textarea>
</div>
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</textarea>
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<textarea rows="30" wrap="soft">
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</textarea>
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</textarea>
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Revision as of 21:55, 19 June 2012

Header

Protocols

Meeting Notes

Pre-April

  • 2/17/12
  • 2/19/12
  • 2/26/12
  • 3/4/12
  • 3/6/12
  • 3/7/12
  • 3/11/12
  • 3/25/12
  • 3/27/12

    • April

  • 4/1/12
  • 4/3/12
  • 4/4/12
  • 4/8/12
  • 4/14/12
  • 4/15/12
  • 4/17/12
  • 4/22/12
  • 4/24/12

    • May-July

  • 5/25/12
  • 5/29/12
  • 6/1/12
  • TBD
  • TBD
  • TBD
  • TBD
  • TBD

    • July-August

  • TBD
  • TBD
  • TBD
  • TBD
  • TBD
  • TBD
  • TBD
  • TBD

    • August-Jamboree

  • TBD
  • TBD
  • TBD
  • TBD
  • TBD
  • TBD
  • TBD
  • TBD

    • Select a date to read respective meeting notes.


    Retrieved from "http://2012.igem.org/Team:UIUC-Illinois/Notebook/MeetingNotes"