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From 2011.igem.org

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

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 F-, Δ(araD-araB)567, Δ(codB-lacI)3, &lambda-, rpoS396(Am), rph-1. This strain will be helpful for us since it is both araBAD and LacI negative. This means we won't have to put an arabinose repressor in our plasmid which will cut down on construction time and allow for more testing time! We also needed the strain to be ∆LacI since we will be testing the Lac repressor and operator.

We also made carbenicillin plates and diagnosed the "bad" carb plates as chloramphenocol plates. Our mistake came when we used an older stock of carb which was labelled only with a 'c'. Lesson learned; don't assume anything!

--Friday--

Transformed all of our LacI mutants again along with E0240+psb1k3. All of the LacI mutants were plated on newly made Carb plates. Hopefully they have the correct antibiotic this time! We also got a new strain, JS006, which we requested from another lab. We'll make glycerol stocks and competent cells of these.

--Saturday-- Took plates out and all had growth=carb plates good! It's amazing what plating on the right antibiotic will do. All of our plates which had failed just a few days ago had colonies with a few of them being red.

Week 4

--Monday 7/04/11--

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.

--Tuesday 7/05/11--

BW22826 Cells had very fast growth overnight, and were too overgrown to use for competent cells in the morning. The procedure was restarted late at night (around 9:30) and lesser amounts of starter culture were used (1, 2, and 2 mL) to ensure the cells don't overgrow. One culture was placed on a shaker at room temperature, the other two were placed on a bench at room temperature.

We inspected our LacI mutants. Most of the plates were covered in white colonies, though a few had some slightly pink colonies, indicating mutations were successful. The cells were plated on IPTG+ plates, so we expect induction; it is possible that the mutation rate was high and the promoter function was destroyed in a majority of colonies. Ten pink and ten white colonies were replated on IPTG+ plates, to rule out poor induction on our initial plates.

E0240 variants with B0034 and with no RBS in pSB1K3 were cultured.

--Wednesday 7/06/11--

The making of our competent cells was successful! We were able to produce two full boxes today, with good amounts of cells in each of the tubes. Hopefully we won't have to make more for a while.

We also miniprepped the E0240 variants from yesterday so that we could proceed with construction. We digested these minipreps along with our three main promoters (R0040, R0051, R0010) so that we could ligate them together.

--Thursday 7/07/11--

Today started slow but ended in a flurry! We ligated three promoters (R0010, R0040, R0051) to E0240 (GFP) and transformed them. We also retransformed our mutant LacI ligations to try and get even more. The previous batch of potential mutants which were different shades of red on a replica plate got replated to see if we'll be able to see a clearer difference between shades of red on streaked plates. So far there appears to be a good range of redness which is a positive sign that our mutagenic pcr worked somewhat well. Out of 12 plates, we got 9 possible mutants. We're planning on taking a closer look with our Tecan plate reader early next week. This will give some more definitive proof of good mutants or not. These mutants also got cultured so we can prepare them for sequencing soon.

In the Dh5α strain, LacI repressor is always present so, upon induction we expected to see red. It seems from our mutation protocol that we mutated them too heavily since most colonies were white even with IPTG present.

--Friday 7/08/11--

Mutated C0012, C0051, C0040. Ligations of promoters to GFP looked green=good! Made control constructs for R0010 Ligated R0040 and R0051 mutants into promoter screening plasmid for screening!

Week 5

--Monday 7/11/11--

While we are making and testing promoter mutants, we started making repressor mutants. We'll need them eventually and the mutagenic PCR's don't take too long to do while things are slow. However, testing out these mutants and finding a good range of repression is the tough part. We also cut the wild-type repressors for construction of our testing plasmid.

--Tuesday 7/12/11--

--Wednesday 7/13/11--

Finally got the Tecan set up how we want it for characterization of our mutants.

Miniprepped J61002 in a chloramphenicol backbone, this should come in handy later!

Completely reorganized the 4 degree room. Now we can actually find our plates!

--Thursday 7/14/11--

Uh-oh! Our transformations from yesterday failed. Colonies on all of the controls! We're going to re-digest the following parts:

• C0040 (Cut with XbaI and PstI)
• C0012 (Cut with XbaI and PstI)
• C0051 (Cut with XbaI and PstI)
• C0051 (Cut with SpeI and PstI)
• E0240 (Cut with XbaI and PstI)
• B0034 (Cut with SpeI and PstI)

Set up PCR reactions in order to Gibson our project together. The parts PCR'd were c0012, C0051, C0040, I13458, I13453, E0240, R0010, R0051, and R0040. Will see how they look on a gel and then Gibson them together. So many primers!

Keegan took the GRE today!

--Friday 7/15/11--

Today was a really fun day! We did some minipreps in the morning but then drove down to UC Berkeley for a NorCal iGEM picnic. We got to talk with our fellow iGEMers from UCSF and Berkeley and got updates on how their projects were going. It was nice to see that there is a community of synthetic biologists in Northern California that we can share ideas and materials with. After a relaxed lunch, we had a small meeting to discuss how to achieve the medal requirements. We discussed helping each other to fulfill one of the gold medal requirements with the Berkeley team since some of our software tools might be useful for their project this year. We then got to see their lab and received some pBAD+AraC since those parts from the registry seem to be faulty. Overall a really good day!

Week 6

--Monday --

We weren't able to get all the parts for Gibson-ing our construct together. We're still waiting on the 3 repressor PCRs and the pBAD promoter part. The other's have been extracted and have decent concentrations for a Gibson mix.

Today we transformed pBAD+AraC for PCRing tomorrow.

We also proceeded with the construction of our testing plasmid. We digested C0040, C0012, C0051 at XbaI and PstI so that we could ligate it into B0034, which we cut at SpeI and PstI. Additionally, we cut all of our promoter-GFP parts at EcoRI and SpeI, so that we could ligate them with I13453, cut at EcoRI and XbaI. We ran them on a gel, purified them, and plan on ligating and transforming them tomorrow.

--Tuesday --

We ligated and transformed yesterday's gel purifications today. if this works, we'll be very close to finishing construction!

--Wednesday --

Strange! Our plates from yesterday are covered with lots of green colonies, even the parts that don't contain GFP! We're going to do some tests; perhaps our media is contaminated, or the water that was used for ligations. We will re-ligate these parts, and re-digest from the minipreps. We ran these re-digestions on gel in order to extract the segment we wanted, but noticed some odd things about the bands. In the lanes containing our vectors, there were three bands where we would expect to only see one. This indicated to us that there was likely some contamination before our digestion. We went ahead and transformed the re-ligated gel purifications from earlier this week.

Took a picture of Nick. He wasn't too happy. We fixed it.

--Thursday--

Once again, our plates are contaminated with green colonies. After some deliberation, we believe that our old media was contaminated with GFP-containing bacteria, and that media was used to culture some of our parts prior to a miniprep. Those minipreps, then, have both plasmids. We're going to re-streak the parts from glycerol stocks, and make new minipreps.

Aaron made the bench into an igloo today complete with dry ice effects.

We also re-transformed C0051+E0240. Let's hope it works this time.

--Friday--

Week 7

--Monday 7/25/11--

We continued with construction today and, once again, extracted the cut Promoters+GFP and repressors from a gel. We ligated the promoters+GFP in from of I13453 and the repressors before B0034. Hopefully this is the last time we will have to ligate and transform these parts.

--Tuesday 7/26/11--

The insert controls for yesterday's transformations looked good, but there were still a good amount of colonies on the vector controls. We decided to do a PCR screen of these parts to make sure that they were the right length.

Today was also "Tip Day"! We filled enough tip boxes to last us a quite a while.

Week 8

Week 9

Week 10

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