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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 6/13/11--

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 6/14/11--

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.

--Wednesday 6/15/11--

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 6/16/11--

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.

--Friday 6/17/11--

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.

--Saturday--

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

--Sunday--

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

Week 2

--Monday 6/20/11--

Today we nanodropped the 12 digested mutated LacI promoters and the J61002 promoter screening plasmid such that we could ligate them:

Sample ID	ng/uL	260/280
LacI 1	188.93	1.19
LacI 2	113.96	1.26
LacI 3	87.29	1.19
LacI 4	88.23	1.20
LacI 5	109.35	1.22
LacI 6	90.64	1.18
LacI 7	536.16	1.33
LacI 8	415.75	1.33
LacI 9	462.50	1.33
LacI 10	544.12	1.36
LacI 11	461.32	1.33
LacI 12	498.83	1.35
J61002	11.92	1.95

We then ligated the 12 mutated LacI promoters into the J61002 promoter screening plasmid. We had planned to transform all of these ligations but unfortunately ran out of LB+Carb plates. We were still able to transform about 7 of the 12 ligations. We also rehydrated and transformed the B0015 terminator.

We miniprepped and digested the following parts:

• I13458 (with EcoRI and SpeI)
• I13453 (with EcoRI and XbaI)
• R0010 (with SpeI and PstI)
• R0040 (with SpeI and PstI)
• R0051 (with SpeI and PstI)

--Tuesday 6/21/11--

After yesterday's plate shortage, we took on the new task of making new LB+Carb plates.

We also nanodropped many of the minipreps (MP) and gel purifications (GP) from earlier:

Sample ID	ng/uL	260/280
J23101 MP	338.75	1.87
I13456 MP	245.92	1.86
I13453 MP	181.48	1.85
R0010 MP	115.98	1.86
R0051 MP	101.21	1.89
E0040 MP	240.71	1.82
R0040 MP	80.18	1.92
C0012 MP	165.16	1.87
R0010 GP	22.42	1.73
I13458 GP	16.63	1.86
R0040 GP	20.39	1.62
I13453 GP	15.10	1.74
R0051 GP	7.27	2.69

--Wednesday 6/22/11--

Miniprepped B0015 and made glycerol stocks of I13453, I13458, and B0015. We also made some 10 mM IPTG from more concentrated stocks.

--Thursday 6/23/11--

After waiting most of the week some of our primers finally came in! We ran PCR with our B0034+E0240 forward primer and our E0240 forward primer.

--Friday 6/24/11--

PCR purified B0034+E0240 and E0240. Digested B0034+E0240 with XbaI and PstI.

Today we digested the following parts:

• B0034 (with SpeI and PstI)
• C0040 (with XbaI and PstI)
• C0051 (with XbaI and PstI)
• C0012 (with XbaI and PstI)
• J61002 (with EcoRI and PstI)
• psB1K3 (with EcoRI and PstI)

Week 3

--Monday--

Today we made Dh5a competent cells as we had run out of our stocks. Keegan made a Gibson volume calculator which will easily calculate the volumes of each part you need when you input the concentrations of those parts to make sure you have equal molar amounts. It will be handy for quick calculations of assemblies of up to 10 parts at a time.

--Tuesday--

Today we transformed the LacI promoter mutants into the screening plasmid Bba_J61002. We're hoping to see red from the RFP that is built into the screening plasmid. Ideally we will see shades of red corresponding to differing strengths of the mutant promoters. Although we're planning on using a fluorescent cell sorter, a cursory screening could be useful just to make sure our error-prone PCR was successful. We transformed into Dh5a E. coli so we had to induce our plates with IPTG since this particular strain is LacI+.

We had some very unusual weather here today. Normal Davis summers are very hot and dry and today should have been around 90-100F but it was rainy and cold! Global climate change perhaps?

--Wednesday--

We realized today that we needed to test some carb plates that we made last week. Our suspicions arose when 16 ligations that had previously been transformed successfully failed to grow on our new plates. We did a quick test by streaking a couple plates with a part in an AK backbone. We streaked the same part on both Kan and Carb plates and only the Kan plates had any growth on them. We will make some new Carb plates tomorrow and hopefully they'll work! Today we also designed the rest of our Gibson assembly primers for constructing our screening plasmid. We'll order tomorrow and in about a week be able to assemble everything fairly quickly. In the meantime we'll continue using biobrick assembly.

--Thursday-- We did PCR of E0240 with and without B0034. We wrote our formal letter to Novozymes and set up digest with E and P of E0240 modified with and without B0034. We also got a new strain of E. coli, BW22826 which is

Made plates and diagnosed bad carb plates as chloramphenocol plates

--Friday-- Transformed all of our LacI mutants again along with E0240+psb1k3.

--Saturday-- Took plates out and all had growth=carb plates good!

Week 4

--Monday-- Happy 4th of July for all the Americans! Today we started making more competent cells for the new strain BW22826. Tomorrow we will complete the protocol.

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

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