The team started the project June 17, 2013, the immediate week following finals week. The team spent the first two weeks deciding on an iGEM project. The first week consists of a high level discussion about different areas of interest.

Each team member prepared 5 ideas, and the team selected the top five ideas. These ideas include a biosensor for common parasites in water, a biosensor for UV damage, a bacteria that fights cavity, bacteria to produce diesel from food oil, and a composite biosensor that incorporates several biosensors.

Each member focused on one idea. The goal for the day was to determine a possible mechanism. The goal for the day was to determine the feasibility of the idea. The team also considered several new ideas such as MSG detection, urine detection, and biofertilizer.

This day was a continuation of the previous day. Emphasis today was to research previous iGEM teams for inspiration and possible mechanisms and biobricks.

We tried to find one more idea that will be feasible. After meeting with advisors, we tried to focus more on the mechanistic details rather than a high level discussion of the project idea. We also took an inventory of the lab space and began sending out sponsorship emails.

After deciding most of the ideas we have come up with are far from feasible, we focused again on previous iGEM teams, hoping to build on a previous team's work. Each team member was tasked with coming up with a new idea for the next week.

The team began working on some lab work such as making different solutions that will be necessary for the rest of the summer. The team also made competent cells and tried to test competency by transforming them. The week ended with much frustration due to a lack of progress in brainstorming and lab work.

The team decided on roles to improve efficiency and team organization. The team made SOB solution and obtained Top 10 cells from Dr. Leonard (one of our advisors). The team also talked quorum sensing of bacteria as a possible mechanism to detect parasites in water.

The team discovered that UV sensing is already done several times by different iGEM teams. The idea may lack novelty. To improve on the biofuel idea, different starting materials are considered, hoping to increase the financial sensibility by decreasing the value of the starting materials.

Today, the team focused primarily on oral health and tried to come up with several ways to attack biofilm formation in the mouth. The ideas that were discussed include engineering V. Parvula to upregulate lactic acid metabolism, introducing the CovR pathway to disrupt biofilm formation, and using the L-Arginine pathway to produce ammonia. The team also created CCMB solution.

The team learned how to make plates (created 40) using CB antibiotic. The team continued to talk about the three ideas, trying to discover anything that will make the project feasible. The primary hurdle is the mechanism to import lactic acid from outside the cell.

The transformation failed, and no cells grew on the plates. The professors pointed out that the upregulation of lactic acid uptake would disrupt the redox balance within the cell, causing the key enzyme lactate dehydrogenase to work in reverse. However, the team has decided that the project for the summer will involve oral health.

As the team goes into the holiday weekend, very few things are working properly and frustration grew!

The team redid the transformation experiment again. This time, all glassware was washed to make sure they are free of detergent. The team prepared for the meeting with professors by trying to flesh out the idea as much as possible.

The cells did not grow once again. The team divided lab space in preparation of the next phase of the project where each member will have more independence. The team is focusing on pH sensor since that appears to be the common mechanism in all of the ideas that were brought up. To add an extra layer of complexity, the team will incorporate the idea of a "dual promoter" into the design.

The team redid the transformation once again. Most of the materials used is store bought to eliminate any possible errors. Prior to leaving for the break, the team is brainstorming on a list of possible mechanisms for the pH sensor.

The team came in just to check for the results of the transformation. Yet again, no cells grew.

July 4th weekend holiday! No meetings.

This week was spent figuring out why our transformations were failing, designing a plan of action in terms of dual-state promoter construction and practicing miniprepping, gel electrophoresis, and gel extraction. We began to design primers.

Over the long weekend, we spent time identifying genes that were activated at high levels due to a pH shift. We focused on the asr (acid-shock rna) gene and the gadA genes. Their promoters should be pH-inducible. Moreover, a past iGem team from Wisconsin-Madison has already isolated the gadA promoter.

Over the long weekend, no growth was shown on the 7/3 transformation, indicating that our SOB was not the problem. Three lab members independently performed a transformation to test competency of cells prepared on 7/2 using a pUC19 plasmid. The transformations were tested on both Cb and Lb plates. Additionally, our TA Jessica independently ran a transformation testing our cells, plates, and SOB.

Success! Our TA has indicated that our cells are competent comparable with her Dr. Jewett’s lab. She realized that the plates we thought were Amp were actually Cm, and this could be the reason why some of our transformations are failing. Our SOB and cells are fine. We also had an advisor’s meeting, where they suggested we use a low-copy plasmid for our construct, create a GANTT chart before next week, assign people to become specialists in each protocol we will be doing over the summer, and to practice these protocols over the next week or so. Afterward, Brendan showed the lab how to make a gel, and he and Nirmal performed a transformation on the correct Amp plates using both the pUC19 and pL plasmids. Viral inoculated the cells Jessica transformed, so we can practice a miniprep tomorrow.

We finally had a successful transformation! The cells containing pL grew on the Amp plates. The pUC19 cells did not grow, indicating that the plasmid, in conjunction with the mislabeled plates, was the source of our transformation errors. We then practiced a miniprep, gel electrophoresis, and subsequent gel extraction.

We spent the entire day in the library, coming up with a schematic for how to construct our dual-state promoters. We struggled particularly with designing how to add spacers so that we need as few primers as possible to save money. Ultimately we decided to have one 50bp sequence with the same restriction enzyme site on both ends. We would add this 50bp sequence repeatedly to create 100bp and 150bp spacers. In the afternoon, we learned how to design primers. We designed a primer together, and then decided that we would go home and design primers on our own and compare the next day. This should help eliminate errors.

We had a meeting with Jessica for feedback on our schematic. She saw a problem with having the same RES on both ends of the spacer, because then the spacer could be added in either direction, and our sequences would not be consistent. Moreover, the sequence is very short, which will be difficult to insert into a plasmid in terms of efficiency. We realized overnight that there was an RES in the middle of the constitutive promoter-RBS-GFP construct we were going to use from Jessica. So we re-designed the schematic to work around this RES using site-directed mutagenesis.

We finished designing primers, met with a representative from IDT who was kind enough to sponsor our team, and began practicing with our Biobrick Kit.

We spent the first half of the day trouble-shooting on our design schematic, confirming the proper sequences of our parts, and then designing promoters one by one together.

We prepared for our meeting with our advisors by drawing a final schematic, and compiling a list of primers. Our advisors informed us that to simplify our design,we should construct primers with two restriction sites. This way we can just use the Biobrick methodology to construct our dual-state promoters. We set to work re-doing our primers in this manner in the afternoon. We also met with a representative from IDT, who was very helpful in providing us with $250 worth or free oligos, as well as t-shirts, lab notebooks, and nuclease-free water.

Jessica informed us that we should be checking the temperatures and GC contents of the portion of the primer that anneals to our target. Before this, we had been checking and adjusting the temperatures of the entire strand. This means we need to re-do all of our primers.

We performed a transformation with pSB4A1 from the kit (Kit Plate 5, Well 21D) to practice transforming a plasmid. Past iGem teams have had trouble properly extracting the DNA from the kit.

The 7/18 transformation did not work. So we ran the same plasmid alongside a positive control using pL plasmid to check our technique.

No Lab Today!

We ran into budget problems today as the primers cost more than the amount of donation we have. We also researched more to change pH in plate media through buffer. Lastly, we worked more on human practices by researching Dr. Zoloth's area of expertise.

We redid the transformation once again. We have controls to make sure that both the plasmids and the cells are good. In addition, we developed a rough draft of a powerpoint for human practices and looked up how to do PCR. We also ordered primers.

The primers arrived today, and we will PCR tomorrow. We also inoculated a colony today. We looked at how to make M9 (minimal media), but we were missing ammonium chloride. This will be postponed until Monday. The transformations worked well, proving again that our cells are competent. Two plasmids from the kit failed, but both plasmids obtained from our Dr. Leonard's lab worked well.

Today we started PCR! We suspended the primers in nuclease free water. We only performed PCR on ASR-RSB. We performed the assay under the guidance of our TA. In the afternoon, we tried PCR purification with last year's kit (our kits will arrive soon!), but the yields were low.

Since our yields were very low, we decided to perform another PCR. This time, we went ahead and used all of the primers we ordered. Afterwards, we purified the DNA with our new kits from Epoch, and the yields were acceptable. Today, we also tried to digest the PCR products from Friday, but due to faint bands, gel purification was aborted. We also transformed cells!

Today, we minipreped pSC101 and ended with acceptable yield. We also prepared digestion on all of the PCR products from yesterday. Lastly, we inoculated the transformed cells in preparation of the miniprep today.

We finished digestion that we started yesterday and started ligation. We have two protocols for ligation (one short and one long), so we will prepare for the longer ligation today. In addition, we minipreped pSB4A5 and got surprisingly high concentration of DNA. Other lab work includes making plates and transforming new cells.

The team finished the ligation protocol and transformed our cells with the products. We will see the results tomorrow. In addition, we started a digestion and ligation cycle of the same parts (ASR-RBS, LPP-RBS, GadA-RBS) today. These are duplicates of the ligation we performed today. We will complete the protocol tomorrow. Lastly, we PCRed all of the primers we have (including new primers that just came in today).

The cells grew! This means the ligation worked since our negative control worked relatively well. We also minipreped these cells to prepare for sequencing. The duplicate ligation prtocol is also completed along with the transformation. The PCR products are also PCR purified.

We completed digestions, ligations, and 12-hr transformations from the weekend. Counted cells for the Lpp-RBS ligations and compared them with the negative control’s. Performed inoculations on 3 colonies from the plate with best ratios, which were 6:1 asr-RBS, 6:1 gadA-RBS, and 1:1 Lpp-RBS.

We miniprepped the inoculations from 8/4. Jessica discussed with us tips to increasing miniprep yields and concentration requirements for sequencing. Samson and Simon completed digestion and gel purification of PCR products from 8/1. Kevin made different acids and buffers for fluorescence assays. Simon made media to make AMP plates.

Performed ligations on the PCR products that were digested and purified yesterday. These include gadA RBS, asr RBS, GFP, Tac RBS, Lpp RBS, asr short, asr long, and gadA. At advisor’s meeting, Prof. Tyo suggested 30ml inoculations and miniprepping to cope with our low copy plasmid.

Transformed ligations from yesterday. We tried doing 35mL inoculations and miniprepping with no avail because the we misused the rotor to spin overnight tubes. We reinoculated the failed ones. Made more SOB and inoculated Top10 cells to make more competent cells.

Samson and Simon digested, digest purified, and ligated cycle 3 redo (from 8/1).

We miniprepped 3 overnight cultures of GFP and digested GFP (1, 2, 3) with X and P, Tac-RBS (1, 2, 3), Lpp-RBS (1, 2, 3) with S and P as well as X and P. We also worked on figuring out the Primer Annealing and Extensions. Afterwards, we ran gels on the digested products and performed PCR purification.

We stopped digesting on Tac-RBS and Lpp-RBS. Samson performed gel purification on GFP digests from 8/19. Viral ordered primers fro 128 bp spacer sequence. Nirmal and Samson ran confirmation gels on the purified digest after PCR of Tac-RBS as well as the digest after inserting into plasmid of Tac-RBS.Brendan performed transformation of ligations of GadA-RBS+GFP and ASR-RBS+GFP.

Ligations of ASR-RBS + GFP and GadA-RBS + GFP worked! Viral miniprepped the overnight cultures of GadA - 9 samples. Nirmal digested 64 bp spacer (8/19 45C) and pSB4A5 (8/9) with E and P. Ran a 60 mL 1% gel. Samson digested Lpp-RBS with S and P and PCR purified. Brendan inoculated Asr-RBS2+GFP2 and GadA-RBS2+GFP2 in SOB and on amp plates.

Nirmal gel purified 64 bp spacer and pSB4A5 from yesterday’s digest. Nirmal then ligated the 64 bp spacer into pSB4A5, with 6:1 and 3:1 ratio. He also transformed. Brendan and Samson prepared GadA-RBS, ASR-RBS, LPP-RBS, ASR-short, ASR-long, GadA, and GFP for sequencing. Viral ran PCR product of 128bp spacer on gel. Then she PCR purified. Samson re-made the M9 media, this time without cassamino acids and with thiamine.

Transformations from yesterday (64 bp spacer into pSB4A5 in 3:1 and 6:1 ratios) did not work. Nirmal believes he did not deactivate at the appropriate temperature. Viral began making competent cells. Nirmal ligated 128 bp spacer into pSB4A5. Samson digested 64 bp spacer and GFP 1, 2, 3 again. Gel purified both digests. Brendan digesting pSB1C3 with E and P. Made digestion master mix.

Brendan PCR purified the pSB1C3 digest from 8/23 and eluting only in 30 uL. Simon and Samson miniprepped weekend inoculations of Asr-RBS+GFP, GadA-RBS+GFP, Lpp-RBS+GFP, Tac-RBS, 128bp spacer, pSB4A5. -Brendan ligated Asr-RBS to pSB1C3, and 64bp (40C, 45C, 50C) to pSB4A5. We inoculated ASR-RBS 1-3, Lpp-RBS 1-5, GadA-RBS 1-3, postive control (GFP plate from jessica), negative control ( just GFP, no RBS), and negative control (no cells). Brendan transformed ligations into cells. Viral miniprepped pSB4A5.

Brendan’s ligations of Asr-RBS + pSB1C3 and 64bp (40C, 45C, 50C) + pSB4A5 looked good in terms of ratios. Diagnostic digests on Tac-RBS, Lpp-RBS+GFP, and 128bp failed. Samson’s inoculations in M9 media did not work. Simon made more Cb (AKA amp) plates. Sequencing results came back. Everything besides Tac-RBS worked! Inoculated Asr-RBS in pSB1C3 and 64bp spacer in pSB4A5 overnight. Viral started 128bp spacer over from the PCR step.

Brendan miniprepped Asr-RBS in pSB1C3. Simon miniprepped 64bp spacer in pSB4A. 5. Samson made Knight M9 media, replacing glucose with glycerol and Coldspring Harbor M9 media. Used Jewett lab material for both media.Samson inoculated LRG , ARG, GRG,Tac (positive), GFP (negative), swirl (negative), nothing (negative) in Knight M9, Coldspring M9, and SOB. Samson and Brendan digested of 64bp. Viral digest LR3 and GFP1 (both sequence confirmed) and ran on gel. Viral also an transformation, testing competency of her competent cells made on 8/26. Simon tested his Cb plates.

Viral’s competent cells didn’t work. Samson’s Knight lab M9 media worked, but Coldsprings didn’t. Simon’s Cb plates worked. Brendan ran the diagnostic digests (again) of 128bp, 64bp, and LRG. 128bp seemed to work, but the other two didn’t. He also inoculated 10 more LRG, in addition to the 5 we already have, for sequencing. We digested GadA with E and S, 64bp with E and X, 128bp with E and X. We redigested LR and G and ligated them. We also ligated the LR and G digests from before. Samson performed fluorescence assays.

We purified digests, ligated, and transformed 64bp & 128bp. We also performed maxipreps. Samson’s redigestion didn’t work. Transformations of Brendan’s ligations didn’t have much difference between positive and negative. Samson worked on fluorescence assays.

We thought the reason for failing digestions was the water. Samson redigested GadA (2, 5, 4) and also digested Asr (short) 2, Asr (long) 3 with old water. Brendan redigested GadA (2, 5, 4) and Asr (short) 2, Asr (long) 3 with new water. E and S were used for the digestions.

Nirmal and Brendan redigesting 64bp (1, 2, 3) and 128bp (1, 2, 3) with E and X with new water. Nirmal ran GadA and Asr (short/long) digests from yesterday on gel but results failed. Nirmal and Brendan digested GadA-RBS with different combinations of old and new restriction enzymes. Samson redid the fluorescence assay. Sequences of LR1a, AS2, AL2, LR2a, and TR2 worked!

Nirmal ran diagnostic gels on the digestions from yesterday but did not work. Samson made new TBE buffer. Nirmal redigested with 1000ng instead of 500ng. Digestions on GadA-RBS worked! Samson inoculated 3 colonies of Asr-RBS in pSB1C3.

Brendan gel purified Asr Short 2, Asr Long 2, GadA 4 digests with E and S from yesterday and maxiprepped Asr-RBS + pSB1C3 inoculations. Samson digested 64bp and 128bp with E and X and PCR purified.

Brendan digested LRG with E and X. Viral made more Cm plates Viral PCRed GadA4, Asr Short 2, and Asr Long 2 and purified. Samson ligated Asr short 2 to 64bp and 128bp and transformed. Nirmal ligated Asr long 2, GadA4 to 64bp and 128bp and transformed. Samson used newly made M9 for inoculation.

Brendan digested more pSB1C3 with E and P. Brendan and Samson maxiprepped Asr-RBS+pSB1C3 for sequencing. Nirmal ligated Asr-RBS, GadA-RBS, Lpp-RBS (all from 8/1) to pSB1C3 (8/26) and transformed. Viral performing diagnostic PCR on Asr long 2, Asr short 2, GadA 4 + 64bp/128bp . Samson worked on fluorescence assays.

We sent in Asr-RBS+pSB1C3 maxipreps from yesterday to sequence. Brendan gel purifying pSB1C3 that were cut with E and P. Viral performed diagnostic PCR of the AL, AS, and G + 64/128. Chose the ones that worked and Brendan miniprepped them. Some worked. AS-128 and AL-128 both didn’t work at all. Viral ran re-diagnostic PCR on other AS-128s and AL-128s. Brendan redigested pSB1C3 with E and P using CIP to de-phosporylate the ends of the pSB1C3 so they don’t re-ligate to the insert. Nirmal performed diagnostic PCR on AR, GR, LR with pSB1C3. Inconclusive.

Brendan nanodropped minipreps from yesterday. Brendan sequence confirmed that ASR-RBS + pSB1C3 3 from 9/6 (sent on 9/10). Nirmal digested. Brendan purifying phosphotased digests of pSB1C3 (E + P).

Nirmal redigested AS2-64, AL2-64, G4-63, GR-128. Viral colony PCRed more AS2-128, AL2-128. Didn’t seem to work. We submitted AR-pSB1C3 2 to iGEM!

Brendan maxipreped weekend cultures of AS2-64, AS2-128, AL2-64, AL2-128, G4-64, G4-128. -Nirmal purifying, ligating, and transforming AS2-64, AL2-64, G4-64, and G4-128 to LRG. Brendan checked sequences of GR 1, 2, 4 and LR 1, 3, 5 that were in pSB1C3. Brendan digesting minipreps from above with E and X, digesting LRG 8 (8/30) with E and S.

Brendan PCR purified products digested with E and X. Nirmal gel-purified products digested with E and S

Nirmal colony PCRed all the dual-state constructs transformed yesterday: (AS2-64)8-LRG 4, (G4-128)2-LRG 1, (AS2-64)2-LRG 2, (AS2-64)8-LRG 3, (AS2-64)-LRG 1, (G4-128)2-LRG 2. Samson ligated and transformed LRG to phosphatased AS-128, G4-64, GR-128

Miniprepped inoculations from yesterday. Digested LRG, AS, AL, G for ligation and transformation tomorrow with no spacer. Colony PCRed transformations from yesterday to see if dual-states worked. None worked.

Digest purified Asr Long 3, Asr Short 2, GadA4, LRG8 1,2,3.-Ligating and transforming AL3-LRG, AL2-LRG, G4-LRG, (G4-64)(2,4,7)-LRG, (AL2-64)8-LRG, (AL2-128)4-LRG, (AS2-128)4-LRG. Digested pSB1C3 with E and P. and ran it on gel.

Gel purified yesterday’s digests