Team:UC Davis/Notebook

From 2011.igem.org

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<h1>Week 1</h1>
 
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Goals: This was the first week that we've all been together in lab to work on the project. Most of our goals for the week deal with getting our work for the summer set up. We need to design the constructs that we are going to build and rehydrate/transform all of the parts needed to build these devices. Because promoter and repressor mutants are critical to our project, we also need to develop a good protocol for mutagenesis. The other thing that we will need to focus on setting up for this week is the our layout for the wiki. It is important that we all have the same vision in mind for how we will present our project.
 
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Progress: This was a very successful and productive week. We are now all settled into our new work bench with all of the space, equipment, and motivation to get a good project going. We've rehydrated and transformed all of the parts that we will need for now. Additionally, we've tested out many different conditions for mutagenic PCR on the LacI promoter (R0010) and will find out soon if it was successful. On top of all of that, our wiki is starting to look pretty good. This week definitely got our summer started off on a positive note!
 
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--Monday 6/13/11--
 
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Today we had our first real day of lab work.  We settled ourselves into the lab bench that we will work at for the summer and set up our area.  Along with familiarizing the new members with lab protocols, we had a productive day of hydrating and [[Team:UC_Davis/Protocols#transformation|transforming]].  We really liked the linearized vectors that the Registry sent along with the plates, very nice addition. Today we designed what our wiki will look like and talked about some finer of the details of our project such as the exact construction method and a work plan.  We are considering using Gibson assembly for all of our construction although in our lab it is still being debugged in a different side project.  Results are promising though as biobricking has its drawbacks.  Among other things, planning of an all-you-can eat sushi lunch is in the works.  We will plan our work schedule around that. 
 
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-Rehydrated parts from 2011 Registry Distribution:
 
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*-R0040=Tet promoter in psb1a2
 
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*-C0040=Tet repressor in psb1a2
 
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*-C0012=LacI repressor in psb1a2
 
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*-C0051=Lambda cI repressor in psb1a2
 
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*-R0051=cI-regulated promoter in psb1a2
 
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*-I732006=LacZ-alpha in psb1ak3
 
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*-R0010=LacI regulated promoter in psb1a2
 
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*-E0040=GFP coding region in psb1a2
 
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*-J23101=Constitutive promoter in j61002
 
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*-C0080=AraC repressor/activator in psb2k3
 
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*-I13458=pC+AraC in psb1a3
 
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*-I13453=pBAD promoter in psb1a3
 
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*-B0015=Double terminator in psb1ak3
 
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-[[Team:UC_Davis/Protocols#transformation|Transformed]] all of these hydrated parts
 
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<h1>Notebook</h1>
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--Tuesday 6/14/11--
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<img src="http://farm7.static.flickr.com/6193/6153279043_debe94f8f9_b.jpg" width="300px" height="200px" align="left">
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Today we tested out [[Team:UC_Davis/Protocols#ER-PCR|error-prone PCR]] on the LacI promoter.  We are adding MnCl2 to a few of the tubes and changing the nucleotide concentrations in other tubes.  We want to test out some different screening techniques for a rapid hunt.  Characterizing thousands, possibly tens of thousands of promoter mutants is quite a daunting task and we want to make it as easy as possible.  We are looking to using a fluorescent cell sorter on campus which could narrow down a few good candidates for more rigorous testing.
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Welcome to our notebook. Here, you may will find summaries of our progress, pictures from throughout the summer, and information on the protocols we use in our lab.<br><br>
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We began work on the actual wiki with some code that I (Tim) wrote over the weekend. It works perfectly in an unrestricted webpage but once I put it in iGEM's wiki, it doesn't work. We'll have to figure this out.   </p><p>
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We cultured all parts and [[Team:UC_Davis/Protocols#digest|digested]] to check on a [[Team:UC_Davis/Protocols#gel|gel]]. All parts checked out.  Sent out all hydrated parts to get sequenced.
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We [[Team:UC_Davis/Protocols#ligate|ligate]]d mutants into screening plasmid to see if our error-prone PCRs had visible mutants.
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Additionally, we rehydrated and [[Team:UC_Davis/Protocols#transformation|transformed]] part C0080 (araC regulatory protein) as we had forgotten to rehydrate it on Monday.
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--Wednesday 6/15/11--
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Today we ordered primers for mutagenesis of LacI promoter, cI promoter and tet promoter. They didn't have adequate overhangs on the forward primer which could make it difficult or possibly impossible to [[Team:UC_Davis/Protocols#digest|digest]] in order to put in a screening plasmid. We also ordered primers to replace RBS (Bba_B0032) in Bba_E0240 to Bba_B0034.  Glycerol stocks were made of our hydrated parts.  We sent all of our parts in to get sequenced to avoid problems we've had in the past with incorrect things from the Registry.   We [[Team:UC_Davis/Protocols#miniprep|miniprepped]] and [[Team:UC_Davis/Protocols#digest|digested]] the parts that were [[Team:UC_Davis/Protocols#culture|cultured]] in order to do a rough check on a [[Team:UC_Davis/Protocols#gel|gel]] while we're waiting for our sequencing data to come back.  
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You may select a week above to travel directly to it, or begin at Week 1 <a href="https://2011.igem.org/Team:UC_Davis/Notebook/Week_1"><font style="color:#EE3333; size:40px;"> here</font></a>.
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If you're interested in what we've been up to since the first jamboree, you can see our progress <a href="https://2011.igem.org/Team:UC_Davis/Notebook/Post_American_Jamboree"><font style="color:#EE3333; size:40px;">here</font><a>.
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We [[Team:UC_Davis/Protocols#miniprep|miniprepped]] and [[Team:UC_Davis/Protocols#digest|digested]] the following parts with XbaI and PstI:
 
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*-R0040
 
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*-C0040
 
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*-C0012
 
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*-C0051
 
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*-R0051
 
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*-I732006
 
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*-R0010
 
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*-E0040
 
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*-J23101
 
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Additionally, GFP (E0040) was [[Team:UC_Davis/Protocols#digest|digested]] with EcoRI and SpeI.
 
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--Thursday 6/16/11--
 
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Today was an exciting day!  We went down to Stanford for a meetup with iGEM teams from UCSF, UC Berkeley, and Standford/Brown. With the little time we had before our road trip, we were able to [[Team:UC_Davis/Protocols#miniprep|miniprep]] C0080 and [[Team:UC_Davis/Protocols#digest|digest]] the mutant LacI promoter. Once at Stanford, we discussed our projects and our strategies for approaching them and talked with each other to share information that could be valuable to each other's projects.  The UCSF team is very impressive being mostly comprised of high school students who seem very sharp.  After a bit of mingling, we all headed to a different part of campus where SB5.0 was happening.  We were lucky enough to be able to attend the poster session which was great!  There were so many posters which all presented interesting things that were similar to things we have worked on or were interested in working on. A few notable posters showed mutating one domain in an efflux pump that were aimed at pumping out biofuel.  One poster which we were interested in was one that raised the idea that DNA could be made with a different backbone which could possibly introduce another mode of isolating synthetic systems in a complex cell. No doubt there would be a boatload of work involved with building artificial machinery to do the basic functions which have evolved for millions of years in nature. Perusing the posters gave us some ideas and really motivated us to do work on our project.
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<h2>Gallery</h2>
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We have a ton of photos in our gallery that didn't end up being used elsewhere on the website, including a few that were too silly to post anywhere else. Check them out <a href="https://2011.igem.org/Team:UC_Davis/Gallery"><font style="color:#EE3333; size:40px;"> here</font></a>.<br><br>
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--Friday 6/17/11--
 
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Today we did some [[Team:UC_Davis/Protocols#transformation|transformations]] of Bba_E0240 [[Team:UC_Davis/Protocols#ligate|ligated]] to our magic screening plasmid.  Those plates will be taken out on Saturday morning and [[Team:UC_Davis/Protocols#culture|cultured]] Sunday evening.  We also ran our [[Team:UC_Davis/Protocols#digest|digestions]] on a [[Team:UC_Davis/Protocols#gel|gel]] to check the lengths.  The mostly came out well.  Some of the promoters were very faint and a bit inconclusive but we'll rerun those later. 
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<h2>Protocols</h2>
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[[File:UCD Gel1.jpeg|frameless|Gel of digested parts.]]
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Looking for the mix for our mutagenic PCR protocol? Performing a ligation and you're not sure how much vector to use? Check out our <a href="https://2011.igem.org/Team:UC_Davis/Protocols"><font style="color:#EE3333; size:40px;"> Protocols</font></a> page for step-by-step instructions on how to replicate our experiments.<br>
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Sushi buffet today.  Fullness has taken on a new meaning.
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--Saturday--
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Today Tim took out the [[Team:UC_Davis/Protocols#transformation|transformation]] plates and put them in the 4 degree room for storage until tomorrow.
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--Sunday--
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Today Keegan cultured all the transformants in order to [[Team:UC_Davis/Protocols#miniprep|miniprep]] them on Monday.
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<a href="https://2011.igem.org/Team:UC_Davis/Notebook/Week_2" style="color:#EE3333; font-size:35px;">Go to week 2</a>
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Latest revision as of 21:05, 28 October 2011

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Criteria

View our judging criteria for iGEM 2011 here.

Week Selection

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Notebook

Welcome to our notebook. Here, you may will find summaries of our progress, pictures from throughout the summer, and information on the protocols we use in our lab.

You may select a week above to travel directly to it, or begin at Week 1 here.

If you're interested in what we've been up to since the first jamboree, you can see our progress here.

Gallery

We have a ton of photos in our gallery that didn't end up being used elsewhere on the website, including a few that were too silly to post anywhere else. Check them out here.

Protocols

Looking for the mix for our mutagenic PCR protocol? Performing a ligation and you're not sure how much vector to use? Check out our Protocols page for step-by-step instructions on how to replicate our experiments.

Retrieved from "http://2011.igem.org/Team:UC_Davis/Notebook"