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From 2011.igem.org

Contents 1 Week 1 2 Week 2 3 Week 3 4 Week 4 5 Week 5 6 Week 6 7 Week 7 8 Week 8 9 Week 9 10 Week 10

Week 1

--Monday--

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 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.

-Rehydrated parts from 2011 Registry Distribution:

• -R0040=Tet promoter in psb1a2
• -C0040=Tet repressor in psb1a2
• -C0012=LacI repressor in psb1a2
• -C0051=Lambda cI repressor in psb1a2
• -R0051=cI-regulated promoter in psb1a2
• -I732006=LacZ-alpha in psb1ak3
• -R0010=LacI regulated promoter in psb1a2
• -E0040=GFP coding region in psb1a2
• -J23101=Constitutive promoter in j61002
• -C0080=AraC repressor/activator in psb2k3
• -I13458=pC+AraC in psb1a3
• -I13453=pBAD promoter in psb1a3
• -B0015=Double terminator in psb1ak3

-Transformed all of these hydrated parts

--Tuesday--

Today we tested out 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.

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. We cultured all parts and digested to check on a gel. All parts checked out. Sent out all hydrated parts to get sequenced.

We ligated mutants into screening plasmid to see if our error-prone PCRs had visible mutants.

--Wednesday--

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 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 miniprepped and digested the parts that were cultured in order to do a rough check on a gel while we're waiting for our sequencing data to come back.

We miniprepped and digested the following parts with XbaI and PstI:

• -R0040
• -C0040
• -C0012
• -C0051
• -R0051
• -I732006
• -R0010
• -E0040
• -J23101

Additionally, GFP (E0040) was digested with EcoRI and SpeI.

--Thursday--

Week 2

Week 3

--Monday--

Today we made Dh5a competent cells as we had run out of our stocks.

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

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