Team Notebook

Week 1

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.

Week 2

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.

Week 3

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.

Week 4

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.

Week 5

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.

Week 6

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.

Week 7

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.