Notebook

Week 1

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

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.

Additionally, we rehydrated and transformed part C0080 (araC regulatory protein) as we had forgotten to rehydrate it on Monday.

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.

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 miniprep C0080 and 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.

Today we did some transformations of Bba_E0240 ligated to our magic screening plasmid. Those plates will be taken out on Saturday morning and cultured Sunday evening. We also ran our digestions on a 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.

Sushi buffet today. Fullness has taken on a new meaning.

Today Tim took out the transformation plates and put them in the 4 degree room for storage until tomorrow.

Today Keegan cultured all the transformants in order to miniprep them on Monday.