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Team:University of Chicago/Meetings
From 2008.igem.org
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Meet with professor Collier and discuss biomaterials applications and other interesting proteins. Also, maybe he knows lots of good stuff about mefp's that we can learn from him! | Meet with professor Collier and discuss biomaterials applications and other interesting proteins. Also, maybe he knows lots of good stuff about mefp's that we can learn from him! | ||
| + | |||
| + | ====Tuesday--July 8--1:30pm--BSLC 216==== | ||
| + | -Niels Holton-Anderson: Grad student with Herbert Waite until very recently<br> | ||
| + | |||
| + | -His thesis was characterizing the proteins that surround the collagen fibers in the mussel adhesive protrusion Of the three proteins 1,3, and 5, foot protein 1 has interesting characteristics | ||
| + | -Highly positively charged | ||
| + | -Thought to be involved with cohesion of proteins in the mussel foot fiber | ||
| + | -Foot proteins are thought to be very unstructured—which is probably good if we’re going to try secretion<br><br> | ||
| + | |||
| + | -Salmonella secretion system requires low metal to be efficiently transcribed. | ||
| + | -However, our foot proteins can bind metals—probably pretty strongly; in fact, this is thought to be why it’s so toxic | ||
| + | - So Nora’s idea is that by expressing the foot proteins, we should be able to induce the expression of the secretion system.<br> | ||
| + | -RGD repeats from collagen have been appended to the end of one of the mussel adhesive proteins, and this somehow improves expression<br><br> | ||
| + | '''What’s the first step?'''<br> | ||
| + | -Get the secretion system and put it in E. coli – waiting for response from supplier<br> | ||
| + | -Generate GFP with Caulobacter secretion sequence | ||
| + | -If GFP is efficiently expressed and secreted, try Mefp-5 | ||
| + | -Order custom Mefp-5 BioBrick – include Flag tag (DYKDDDDK) | ||
| + | -Subclone into IPTG inducible BioBrick expression vector | ||
| + | -How to characterize expressed protein? | ||
| + | -Probably just crude protein prep, treat with tyrosinase, check stickiness | ||
| + | -Then, we’ll add a secretion sequence to the Mefp-5 and try to secrete it in the E. coli | ||
Revision as of 20:48, 8 July 2008
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Meetings
Hokay, so here's the easiest way I could imagine us keeping track of all our various meetings/appointments: a calendar page. I think it would be pretty easy to just list things in order of most recent first, along with who should be there and a brief description of what the meeting is about. You should also post anything important resulting from the meeting after it happens. For instance:
Monday - 26 May 2008 - 1:00PM - GCIS
Who should be there: Everyone. We'll be discussing the papers Steve sent out on May 19 (titled "Concepts"); hearing about E. coli secretion systems (Nora, Thomas); hearing about the Schultz method (Jata); and going over funding (Laura) and infrastructure (Jata, Damon).
Tuesday - 27 May 2008 - 12:30PM - C-Shop
Who should be there: Jata & Damon. We'll be discussing infrastructure, and our pending meeting with Dr. Schonbaum.
Sent Dr. Schonbaum an email:
Dr Schonbaum,
Thank you for agreeing to meet with Damon and I tomorrow in regards to our
synthetic biology research over the summer; we're very grateful for your
help. We're writing you this email to give you an idea of the things we'd
like to cover when we see you.
1. Access: Over the summer, how will we get into BSLC, when and
where are we allowed to go, who can we ask for access to things we need in
areas we cannot go, and what do we need to do to obtain the keys/ID
necessary to make things run smoothly?
2. Supplies: What supplies will we have regular access to, which
need to be ordered, how do we order them, where do we store them, and how
much do you recommend we keep in stock to perform the methods listed
below? What can we do to ensure a smooth transition from summer research
to continuing the research during the next school year?
3. Waste: safety, legal, and environmental concerns?
4. Equipment: what should we get;,what's entirely ours and what will
be shared, will we need special access to the shared equipment if it's in
another lab or operated by someone else (e.g., autoclave, microscopy,
refrigeration)if we really need something that they won't give, how should
we request or buy the equipment?
5. Safety: training, certification, paperwork; rules and where to
get them?
Here is a link to our proposed methods:
https://2008.igem.org/Team:University_of_Chicago/Project
We are working on beginning a collaboration with Phillip Messersmith
(Departments of Biomedical Engineering and Materials Science and
Engineering, Northwestern), who will hopefully assist us in the last
(mussel foot protein specific) steps of our procedure; however, we'd like
to figure out what can be done here and what must be done elsewhere.
Thank you, and we look forward to meeting you tomorrow at 11:30AM!
-Parijata Mackey and Damon Wang
Wednesday - 28 May 2008 - 11:30AM - BSLC 215
Who should be there: Jata & Damon. We'll be meeting with Dr. Schonbaum to discuss lab infrastructure for the summer.
Dr. Schonbaum will email us, but from memory,
- Basically no safety or legal issues
- All the usual molecular biology equipment and reagents will be available
- Officially in Rm 214 (?) but have 24hr(?) access to equipment in the neighboring labs and to reagents in Rm 209
- Keys to Rm 214 and Rm 209 for as many of everyone as Marcia has keys
- Schonbaum can give us His-tagged E. coli vectors
- Schonbaum teaches an all-day lab class for high school students beginning 23(?) June, so we should figure out what we need before then
- Schonbaum will meet us weekly until this works out.
- We have to figure out what we're doing to know what we need. We should do that soon.
Next actions:
- Figure out what we're going to do. ^_^
Friday - 30 May 2008 - 2:30PM - CIS E125
Who should be there: Damon, meeting Sean Crosson to discuss Caulobacter as an expression vector
Crosson has all the plasmids and the Caulobacter and the experience, and will share it all; he has not worked with RsaA secretion personally but he hears it's robust. The reported ridiculously high transcript strength is due also to the stability of the mRNA, not just to strong expression, and that stability may not be conferred to a signal-target fusion mRNA. Caulobacter is really easy to culture and electroporate, and secretes a scum of solid protein that can be skimmed off the top of the culture medium, but that scum has the target covalently fused to a signal sequence. Crosson thinks expression and secretion will be trivial, but cleaving off the secretion signal might take more work.
Next actions:
- Find some way to make available on this wiki the protocols Crosson photocopied for me
Sunday - 01 June 2008 - 11:00AM - GCIS
Who should be there: Everyone.
Wednesday - 04 June 2008 - 6:00PM - GCIS
Who should be there: Everyone. Grab some dinner and head over to this meeting; it will likely be the last one before finals. We need to get some solid blueprints to present to Schonbaum on Thursday, on the following:
Thomas – Bs (Express/secrete mfps)
Nora – Ec (pirating salmonella type III; express/secrete mfps)
Jata – Sc (Express tyrosinase and chaperones, localize to golgi)
Laura – tyrosinase/chaperone exp
Rob – quorum sensing in Bs, Ec, Sc (plausible pathways)
Dan, Ioana - ? (what do you want to work on; we think you're best fitted with Rob's quorum sensing)
Damon - Caulobacter? (Damon, we need to figure out if/how Caulobacter will fit in. See email)
Wednesday - 25 June 2008 - 9:30PM - Rob's science pad
Welcome back everyone! Introduced Jim and Soren, two lab school students who'll be helping us out over the summer.
-Rob contacted Dr. Collier. His research deals with using bio-materials for tissue repair, and he's worked with Professor Messersmith. Says that expression of Mefp in E. coli is something of a lost cause, but we don't know if he's thought of secretion yet. We'll find out when we meet with him Friday.
-Nora contacted Cheryl Nickerson, whose lab recently transferred the SPI-2 island into non-pathogenic E. coli. Promised to put us in contact with Dr. Wilson who did the work, and help us out with protocols and supplying us with the plasmid. Entire group expressed undying gratitude.
-Potentially do some volunteer work with the high-school program RIBs. Presentation on Synthetic biology, verify the coolness of lab work. That kind of stuff.
-We'll have parallel projects going with E. coli and caulobacter, since we already have a lot of information for them.
Next Week's Goals
-Go through the iGEM tutorials. Verbatim. Practice prepping the biobrick plasmids.
-Damon, Soren, and Jim will get a sterile bench tutorial under the tutelage of the lovely Jata.
-Need to make competent cells and media for TOP10/XO1 blue
-Damon will work on getting caulobacter to secrete GFP as a practice.
-Come up with cloning protocol -Need to prep ligation vector with GFP. Design construct with Nora.
-We'll see if caulobacter is really as robust as Professor Crosson says it is.
-Nora will work on expressing the SPI-2 secretion system. Test for expression it using Western blot with anti-SseB antisera.
-Design mefp codon optimized gene.
-Many of us can come up with glue sequences. Compare and contrast always a good idea. -Make a stronger glue!
-Check we have all the supplies we need.
Future meetings
-Everyone will have to attend the PCBio meetings Thursdays at noon. There will be free food. Rob and Damon exempt during BSCD meeting days.
-Have our iGEM meetings before the PCBio meetings
-Tuesdays noon, keep it down to an hour.
Summer will be awesome!
Friday - June 27 - 11:00 am - Abbott 552
Meet with professor Collier and discuss biomaterials applications and other interesting proteins. Also, maybe he knows lots of good stuff about mefp's that we can learn from him!
Tuesday--July 8--1:30pm--BSLC 216
-Niels Holton-Anderson: Grad student with Herbert Waite until very recently
-His thesis was characterizing the proteins that surround the collagen fibers in the mussel adhesive protrusion Of the three proteins 1,3, and 5, foot protein 1 has interesting characteristics
-Highly positively charged
-Thought to be involved with cohesion of proteins in the mussel foot fiber
-Foot proteins are thought to be very unstructured—which is probably good if we’re going to try secretion
-Salmonella secretion system requires low metal to be efficiently transcribed.
-However, our foot proteins can bind metals—probably pretty strongly; in fact, this is thought to be why it’s so toxic
- So Nora’s idea is that by expressing the foot proteins, we should be able to induce the expression of the secretion system.
-RGD repeats from collagen have been appended to the end of one of the mussel adhesive proteins, and this somehow improves expression
What’s the first step?
-Get the secretion system and put it in E. coli – waiting for response from supplier
-Generate GFP with Caulobacter secretion sequence
-If GFP is efficiently expressed and secreted, try Mefp-5
-Order custom Mefp-5 BioBrick – include Flag tag (DYKDDDDK)
-Subclone into IPTG inducible BioBrick expression vector
-How to characterize expressed protein?
-Probably just crude protein prep, treat with tyrosinase, check stickiness
-Then, we’ll add a secretion sequence to the Mefp-5 and try to secrete it in the E. coli
