Team:Jefferson VA SciCOS/Notebook

October 2013 – January 2014

We started raising awareness of the iGEM competition in the TJHSST community by discussing the competition during weekly meetings of Synthetic Biology Society and delivering lectures on basic concepts of synthetic biology, such as transcription and translation, synthetic biology terminology, and methods of assembly to construct a part. We required that all members of Synthetic Biology Society who were interested in joining the iGEM team submit a proposal for the 2014 project by December 15th. We selected six of those proposals for advanced consideration and invited the members who submitted the proposals to join the 2014 iGEM team. We also received the One Question Grant during November, for which we had to conduct an outreach program.

January 2014 – February 2014

We finalized the idea for our project this year: the construction of a 4-node bacterial computer that solves the Traveling Salesman Problem. We reviewed about ten ideas submitted by members of Synthetic Biology Society, which dealt with topics as diverse as an insulin regulation system for the human body and a ciguatoxin poison attenuator, before we settled on this idea. We felt that it was the most feasible project to complete with the resources in the TJHSST lab and provided abundant opportunities to backtrack if a component was not functioning properly. Moreover, due to the combinatorial nature of the problem we were attempting to solve, we believed it had the most potential in terms of mathematical modeling. We also received a generous donation of $1000 from Dr. Brian Becker of Precision Economics, LLC during the month of February.

February 2014 – March 2014

As part of our outreach program, the Mini-Jamboree, we conducted prep lab sessions during meetings of Synthetic Biology Society. The prep labs were intended as a way to introduce freshmen and sophomores to the Biotechnology Lab and to basic research techniques so that they may be able to formulate their own research ideas with ease. The prep labs were based on the protocols that we received from iGEM headquarters. Although the end goal of the prep labs was to create a part that fluoresces red through the transformation of control RFP, we were not able to do the restriction digest or ligation with the participants due to the erratic weather on the east coast this year. Instead, we first taught the students antiseptic techniques, how to grow bacteria on plates, and how to make these plates using agar powder, distilled water, antibiotic solution, and an autoclave. As we neared the end of March, we taught them a transformation protocol using competent cells we had already purchased from New England Bio Labs.

April 2014

4/3/14 – We received 5-alpha Competent E.coli, a BioBrick Assembly Kit, a Q5 High-Fidelity 2X Master Mix, and a Quick-Load Purple 2-Log DNA Ladder from New England Bio Labs. These items were distributed free of charge to all high school iGEM teams that requested them as part of a one-time deal from New England Bio Labs.

4/5/14 – Techstravaganza outreach event at TJHSST. We had two booths: candy DNA and banana DNA extraction

4/9/14 – We transformed wild-type GFP (BBa_E0040) with competent cells. These were cells that were made competent by Dr. Burnett the previous day, so she was not sure whether they would work or not. However, since this was just a trial transformation to familiarize ourselves with the procedure, we decided to use the old competent cells instead of spending time to make our own. We could not use the cells we purchased from the vendor because we thought they were electrocompetent (however, they were not, we just got confused due to the many boxes of cells that belonged to researchers in the Biotechnology Lab that were cluttering the -80 freezer at the time). Due to time constraints, we used the “What a Colorful World” lab procedure that is taught in an introductory DNA science class at our school. This procedure is a simplified version of the complete transformation procedure and does not yield very high efficiency. It was developed by previous iGEM teams but was highly tailored to a specific project, so it is plausible to assume that the same procedure would not work properly for all projects.

4/10/14 – We checked to see if the previous day’s transformation had worked. As expected, we did not see any colonies that fluoresced green under the UV pen light, although we saw a few normal colonies under natural light. Again, we did this transformation mainly to familiarize ourselves with the basic transformation procedure and not to derive results that could potentially be useful for our project.

4/11/14 – 4/20/14 – Lost a substantial amount of time in the middle of April due to spring break. We did do some advance planning for the project during this time however and ordered the DNA invertase Hin tagged with LVA (BBa_J31001) part from iGEM headquarters.

4/22/14 – Received the DNA invertase Hin part that we ordered from iGEM headquarters today. Fast service!

4/23/14 – We did another transformation with an RFP (BBa_J04450) part from 2012 Kit Plate 1, Well 1A. This time, we used the 5-alpha competent E.coli that we purchased from New England Bio Labs. We utilized the protocol that was provided with the purchased competent cells with 5 ul of resuspended DNA from the plate. We plated the transformation mixture on two separate plates – one without a serial dilution and one with a 1:10 serial dilution.

4/24/14 – We checked the results of the previous day’s transformation. We found that the transformation without the 1:10 dilution worked. However, we only observed one transformed colony.

4/30/14 – We transformed three more RFP (all were BBa_J04450) parts from 2012 Kit Plate 1, Wells 3A, 5A, and 7A. We followed a few suggestions made by the protocol that we realized we had not abided by last time. For the previous transformation, we had neglected to set the incubator to shake. This time, we made sure to set the rotation speed per minute to at least 205 (the protocol recommended 250 but we could not use the full amount because it was making too much noise and was beyond the amount regulated by the lab). We hoped for more colonies this time considering that we had neglected to rotate the transformation mixtures last time and had still observed one colony.

May 2014

5/1/14 – We checked the results of the last three transformations. We were not very pleased with the results – while we expected to see more colonies after rotating the transformation mixtures at 205 rpm, we actually got no yields for any of the parts. After consulting with our mentor, Dr. Burnett, we came to the conclusion that our SOC media was contaminated. We did not store it at 4 degrees Celsius after opening it as per the storage directions. As a result, the SOC media became very cloudy over time, indicating the presence of contaminants within the media. Before doing more transformations, we replaced the contaminated media with a fresh vial that we found within other competent cell kits in the lab.

5/7/14 – Before proceeding further with any experimentation with regards to the actual project, we used this as a planning day to figure out when each procedure would be completed and by whom.

5/8/14 – This was the official first day of project. We transformed paths HPP-A0 and HPP-A1 in order to see if the system is working using the 5-alpha competent E.coli procedure that we had used previously without a serial dilution.

5/9/14 – We checked the results of our transformations. The transformations did not appear to work again, even after we seemed to have mastered the transformation protocol. After researching the paths in more detail and reading through the procedures of the Davidson/Western Missouri iGEM team, we realized that the promoter used in the paths was receptive to T7 RNA polymerase. Therefore, our solution was to use purchased T7 Express E.coli cells as they had done.

5/12/14 – Submitted a purchase order for T7 Express E.coli cells, PstI restriction enzyme, and PCR Mix Superfidelity. Throughout this week, we were also in the process of ordering the PCR primers for our new part (although we ran into a bit of snafu with our school’s finance office in doing so that delayed their arrival). Since we were waiting on the T7 Express E.coli cells in order to retransform the pathways, we decided to isolate the plasmid DNA from the bacteria containing the DNA invertase Hin part that we received on a stab culture last month. We streaked ampicillin plates with the bacteria from the stab culture and grew the plates overnight in the incubator at 37 degrees Celsius.

5/13/14 – We made a cell culture for DNA invertase Hin.

5/14/14 – Instead of doing the miniprep, we worked on a grant application because there was not sufficient time in the lab to finish the procedure. Consequently, we had to remake the cell culture and do the miniprep on Friday.

5/15/14 – We made another liquid culture for DNA invertase Hin.

5/16/14 – We performed a miniprep in order to isolate the plasmid DNA containing the DNA invertase Hin part. We were not able to quantify the DNA on the same day due to lack of time, so we stored the DNA in the -20 freezer for later use. In addition, we received all the items that we submitted a purchase order for on Monday.

5/20/14 – We transformed HPP-A2 (BBa_I715044), HPP-A2 + Hin (BBa_I715051), and hixC (BBa_J44000) parts using T7 RNA Polymerase cells that arrived last week. We checked the concentration of the DNA invertase Hin plasmid DNA using the gen5 quantification system

5/22/14 – We checked the transformation results and they were a bit disheartening. We expected to see a few yellow colonies interspersed with red and green colonies for A2 + Hin, but we didn’t see that. The efficiency of the new cells was obviously much higher because there were several colonies. It might have been possible that the DNA in the 2012 plates wasn’t of high quality because it was over a year old, and since that time, our school has been undergoing renovation. The DNA might have been subjected to various climate extremes due to the construction and the many days off school we got from the erratic weather. We reordered all of the parts from iGEM headquarters to make sure that the fault wasn’t the quality of the DNA itself. We also decided to remake all of the ampicillin plates that we had been using because it is possible for the antibiotic to degrade over time, meaning that the bacteria growing on the plates might not be carrying the plasmid containing the part/antibiotic resistance gene at all.

5/23/14 – We made new ampicillin plates. Ratio of antibiotic to agar – 1ul/ml

5/28/14 – We gave a presentation on our synthetic biology outreach efforts at tjSTAR, TJHSST’s annual symposium to advance research, as part of the One Question Grant we accepted at the beginning of this year. During the presentation, we talked about some of the activities we conducted to promote greater awareness of biotechnology and hands-on learning through the Mini-Jamboree, Prep-labs, and Techstravaganza event.

5/29/14 – We received HPP-A0, HPP-A2, and HPP-A2 + Hin from iGEM headquarters. The other parts did not grow in the quality control tests. We also received Primers A and B (forward and reverse primers for first gene half of BFP).

5/30/14 – Slivoskey Grant Presentation. We received Primers C and D in the morning and plated the parts we received from iGEM headquarters on both the old and new ampicillin plates. We also performed a PCR reaction for both gene halves using the primers.

Primer A - 17.87 nmol Primer B - 17.97 nmol Primer C - 14.29 nmol Primer D - 16.31 nmol

Used 1.4 ml to resuspend the DNA. 1ul of forward and reverse primer was used, while 3 ul of the template DNA was used. The 1 ul of each was diluted in a 1:3 ratio.

June 2014

6/2/14 - We checked how the parts we plated on 5/30 looked. Under the UV light, it seemed that colonies on HPP-A0 glowed a distinct yellow color, which indicated that the transformation had finally worked. We also got colonies for HPP-A2 that did not glow without Hin, which was what was desired. We therefore decided to use the HPP-A2 path to construct our composite pathway, and were hoping to do a gel of HPP-A2 to confirm that it was properly constructed if we had the time. (Insert pic of HPP-A2 here)

6/3/14 – Cell culture for hixC

6/4/14 – We purified the PCR products using a PCR purification kit and performed a miniprep for hixC. We checked the concentration of the isolated plasmid DNA on the same day and got a very poor concentration. This was due to 2 reasons – not all of the cell culture was used for the miniprep (only 1 ml of it) and there was a mix-up on the xg and rpm units. The PCR purification protocol and the miniprep protocol used two different units but both used the same microcentrifuge. Whoever was doing the miniprep did not change the units after the person who did the PCR purification changed them initially.

6/5/14 – We decided to redo the hixC miniprep and quantification. We made another cell culture for hixC.

6/6/14 – We finished the miniprep for hixC.

6/10/14 – We quantified the plasmid DNA for hixC and the two PCR products

We did two restriction digests (accidentally messed up the first one by not adding a restriction enzyme to one of the reaction tubes). We cut the hixC plasmid with EcoRI + XbaI and the first PCR product (result of PCR for A and B primers) with EcoRI + SpeI.

6/11/14 – We tried to do a transformation for RBS of moderate strength (B0034) and two terminators (B0010 and B0012). Due to time constraints, we had to push this back to the next day. Instead, we completed a ligation for Product 1 + hixC. Unfortunately, the thermal cycler wasn’t functioning properly on this day. The lid kept on opening, but our sponsor was able to monitor it for the most part. However, we had to keep it at room temperature overnight and then denature the enzymes at 70 degrees Celsius the next day, which may have influenced the efficacy of the reaction.

6/12/14 – We redid the failed transformation of RBS and the two terminators. After discussing the project with our mentor, we realized that it is not practical to do a restriction digest on a ligated part, and thus, had to revise our plan for the rest of experimentation.

6/13/14 – Without wasting another day, we redid the ligation from two days ago. We incubated the reaction tube at room temperature (couldn’t get the waterbaths to 16 degrees Celsius and the thermal cycler wasn’t functioning properly) and denatured the enzymes at 70 degrees Celsius. We transformed both the old and the new ligation mixtures that same day.

6/16/14 - We checked the results of the transformations (the cells grew perfectly!) and made cell cultures for P1 + hixC, RBS, and HPP-A2

6/17/14 - We did a miniprep for the three parts and checked the concentrations of the isolated plasmid DNA using the Nanodrop machine. We got very high concentrations this time, which indicated progressive improvement in the execution of techniques over time.

Subsequently, we did a restriction digest of the three parts as well as the second product of the PCR reaction. P1 + hixC was cut with SpeI and PstI, Product 2 was cut with PstI and XbaI, RBS was cut with XbaI and PstI, and HPP-A2 was cut with SpeI and PstI. Finally, we did two ligations, one of the P1 + hixC part and the second product, to form the composite P1 + hixC + P2 part, and another of the HPP-A2 part and the RBS to form HPP+RBS.

6/18/14 - We transformed the ligation mixtures of P1 + hixC + P2 and HPP-A2 + RBS using T7 Express E.coli cells.

6/19/14 - We made cell cultures of the transformed parts. However, the media was partially cloudy, which indicated the presence of contamination. We wanted to remake the LB broth to ensure as little contamination as possible, but we did not have the time to do so.

6/20/14 - We did a miniprep of P1 + hixC + P2 and HPP-A2 + RBS. We did a restriction digest for both of them by cutting P1 + hixC + P2 with XbaI and PstI and HPP-A2 + RBS with SpeI and PstI. Lastly, we ligated these two parts together, to get our final pathway: HPP-A2 + RBS + P1 + hixC + P2. This was the last day of experimentation for us because our mentor was not available to supervise us after this date. We finished the composite pathway, but we could not test it through a transformation and affirm that all components were constructed properly by running a gel.