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Post-Regionals

Journal

• Defining Objectives
• Techniques Workshop
• Bioinformatics & qRT-PCR
• Sensory Element Fishing
• Electrochem - CPR Oxidation
• Electrochem - Reporter Gene
• Pseudomonas Tools
• Microalgae Tools
• NA Viability Assays
• May - July Group Journal

Protocols

Safety

Project Participants

Bjorn Nikolas Hornbruch (Niko), Felix Chung

July 8-14, 2011

Author: Niko and Felix

This week, details of the latest testing protocol for the product were examined. Different voltammetric methods were compared and researched.

Felix and Niko tried to take more control measurements, but the equipment broke down. We spent Tuesday and Wednesday trying to fix it, but none of my attempts were successful. Afterwards, we tried the wave function generator and oscilloscope, and got the equipment from Dr. Nygren on Thursday. We had some start-up difficulties - more on this to come.

Next week, our main objective is to develop a working test apparatus and take control measurements on tap/distilled water and TPW. We will continue to evaluate voltammetric methods for quantifying analyte concentration in contaminated solution.

July 15-22, 2011

Author: Niko

On Monday Felix and I met with Jason, Maggie’s husband, to talk about electrochemistry. He was able to give us a lot of tips on how to design the experiment. Specifically he recommended that we use a potentiostat to take the measurements. Unfortunately we weren’t able to borrow one from him or Dr. Nygren, so we decided to built our own from op-amps and resistors. On Thursday we acquired the necessary components for the potentiostat.

The plan for next week is to finish building the potentiostat and take control measurements to make sure it is working properly. If not we will have to trouble shoot. Once it is working properly, we will proceed to take measurements with CPR in buffer and eventually in TPW.

July 23-29, 2011

Author: Niko and Felix

This week was spent optimizing the potentiostat and testing its response in lab conditions using cyclic voltammetry. The potentiostat was tuned to the point where 10x amplification of the input signal was possible with very little noticeable bleeding or noise. The output response did not match literature values; we think the problem might be related to our choice of electrodes. The potentiostat was constructed using less than $10 of equipment using operational amplifiers.

n the coming weeks, the testing procedure will be optimized to account for non-ideal testing conditions, as currently, the system only functions in ideal conditions. By ideal conditions, we mean a simple solution of CPRG in water; by non-ideal conditions, we mean CPRG in dirty water. Neither of these "conditions" includes naphthenic acids.

Date: July 23-29, 2011

Author: Stephen Dixon

Saeed performed a test on E.coli carrying the I0500_B0034_I732005 (arabinose promoter followed by LacZ reporter) to measure the conversion of CPRG into CPR. The experiment was at a pH of 7 which was measured at the end. But it certainly wasn't 7 at the start - we assumed that the e.coli would have produced their own buffer and increased the pH with it. At different pH's though, the solution has different optical density because of the indicator (CPR) used in the experiment. So the results may not be indicative of the level of CPR production over time. The results are below

Time(hours)	Optical Density Reading
0.0	0.063
0.5	0.174
1.0	0.336
1.5	0.582
2.0	0.709
3.0	1.381
4.0	1.282
5.0	2.029
5.5	2.447
21.0	2.797

The above table shows the time in hours and the corresponding optical density when measured with light with a wavelength of 535nm.

July 30 - August 4, 2011

Author: Niko

I spent Tuesday morning catching up on the lab work after coming back from vacation. The potentiostat I had built before I left seemed to work like expected, but we still needed to do more testing. On Tuesday and Wednesday we tested it with water and CPR solutions. At first we used copper wire as electrodes, that didn’t work. Since platinum and palladium electrodes are too expensive we decided to go back to carbon (graphite) for working and counter electrodes and Ag/AgCl for the reference electrode. Thursday and Friday were spent installing the software. It took so long since there were compatibility problems between the software and my operating system. As a matter of fact I still can’t access all features of the software. Felix and I also built a device to hold the electrodes in solution so that we would be able to take measurements easier.

Next week in one word: troubleshooting. I need to fully install the software, and my computer seems incapable of this. The next idea I will try is to borrow Stephens laptop, which has an older operating system, and try to install it on his machine. I also have a meeting on Monday with a potential sponsor. Most important, Felix and I will try to produce the characteristic waveform for CPR that we found in literature.

August 5-11, 2011

Author: Niko

I spent a lot of time trying to get the National Instruments software and hardware to work. On Wednesday I got it running on Stephens laptop (the installation and updating took way longer than expected, due to the age of the laptop). I also discovered a possible workaround for using the old setup with the new software that might work using my laptop. But we did not need to try the workaround, because on Thursday we borrowed a potentiostat (including software, cables, etc) from Hamidreza Shahbaazi (He works in the same lab as Maggie’s Husband, who set up the meeting), so the old setup does not need to be used anymore. This is working and Felix is currently conducting experiments.

For next week I will help Felix conduct further experiments. We might have some downtime if we need to wait for ordered chemicals. During that time I can help out other people.

August 1-5, 2011

Author: Felix Chung

Accomplishments:

1. Built graphite-graphite-AgCl cell that responds electrically, but not electrochemically yet.
2. Studied a lot of theory from Quantitative Chemical Analysis (Harris 2007)
3. Characterized our laboratory electrochemical cell digitally.

Results

Our cell responds to electrical signals as a passive element with a significant (+/- 10%) amount of noise. Currently, it appears that redox processes occur at too slow a rate to be detected with our system. A lot of studying must be done to catch up my knowledge of electrochemistry to what is required for our project.

August 8-12, 2011

Author: Felix Chung

Accomplishments

1. Met with several experts at the Briss lab regarding our cell and learned a lot about the expected electrochemical properties of our cell. Acquired a lab-grade potentiostat along with glassy carbon for use as electrodes.
2. Worked with the potentiostat to characterize all non-oxidative systems. Placed an order for zinc phthalocyanine for electrode modification, which is required to observe bulk oxidation of CPR.

Results

Our meeting with experts in electrochemistry led to many breakthroughs. Although we are now held back by lacking a chemical catalyst that we previously didn’t know we had to use, we are now able to compare our constructed potentiostat to a professional one. Our electrodes were found to be insufficient for our desired purposes. We not only acquired better electrode materials, but we also were advised to modify our electrodes with zinc phthalocyanine complexes in order to detect our analyte with sufficient accuracy.

August 12-18, 2011

Author: Niko

On Monday had a meeting with Dr. Rosehart (Head of the Electrical Engineering department) about potential sponsorship from the Electrical Engineering department. He approved with the condition that we also get funding from Biomedical Engineering, so I set up a meeting with Dr. Vigmond (head of Biomedical Engineering) who also agreed to fund us.

The experiments Felix and I were working on had to be halted due to the fact that our chemicals hadn’t been ordered due to software problems at the university. I spent most of the week creating software for the National Instruments hardware, so that we can use our DIY potentiostat. Since I had to learn how to program with the specific tools it takes a while and the work will have to be continued next week.

August 13-19, 2011

Author: Felix Chung

Accomplishments:

1. Changed our constructed potentiostat to implement a digital A/D converter instead of a purely analog setup. We are in the middle of dealing with cumbersome software issues involving this converter. I am continuing to use the lab potentiostat while Niko figures out the software.
2. Continued characterization and testing of blank solutions with the lab potentiostat.

Results:

Along with Niko, we modified our potentiostat to incorporate some changes suggested by Dr. Nygren, including the incorporation of a computer-controlled A/D converter. There are persistent software issues involving this converter, which Niko is trying to sort out. Meanwhile, I’m working with the lab potentiostat and trying to pull as much data as I can that doesn’t require the modified electrodes.

August 19-25, 2011

Author: Niko

This week was spent trying to finish the national instruments program. I got help from Dr. Nygren and tried using example programs, but it still proved challenging. I did manage to find examples that were very close to what we needed online, but it took me until Friday to modify them into three files that together do what we need; one to record data and save it, one to open and display the saved data, and one to generate a saw-tooth wave output.

The plan for next week is to combine the recording/saving and generation files into one file for easier data recording. I also have a meeting with Dr. Gendron and Dr. Cowe Falls on Thursday.

August 22-26, 2011

Author: Felix Chung

Accomplishments:

1. Began mathematical analysis of sample oxidation data.
2. Examined the cost efficiency of printed circuit boards for final prototype.
3. Began cost analysis of final product.

Results: Because our electrode modification compound didn’t come in yet, work on the final phase of our potentiostat remains stalled. In the meantime, I began analyzing different oxidation curves from literature and determining trends for different rates of reaction. It is important for our final product to be able to distinguish positive test results from false positives, background noise, etc.

August 26–September 2, 2011

Author: Niko

Felix and I are still waiting for chemicals to arrive before we can conduct further experiments. They are ordered, but haven’t arrived yet. I finished combining the programs into one. Since that is done, Felix and I are ready to continue with our experiments as soon as the chemicals arrive.

Robert and I met with Dr. Gendron (the Dean of Engineering) and Dr. Lynne (the Director of Students). The meeting was mainly about raising awareness of iGEM in the faculty of engineering and to gain support for future teams and the club. On both accounts this was successful. We got many tips from Dr. Lynne about how to improve iGEM. One of them was that we could apply for the local Leadership Program at the university.

My plan for next week is to help out people while I wait for the chemical to arrive.

August 29 - September 2, 2011

Author: Felix Chung

Accomplishments

1. Implemented a differentiator element on our prototype potentiostat.
2. Tested the functionality of our potentiostat in bacteria-free conditions.
3. Began comprehensive breakdown of circuit elements and functionality.

Results:

This week was spent making modifications and improvements to existing processes. A differentiator element was added to our prototype, which automatically takes the time derivative of the input signal. This will let us easily determine whether or not there’s a oxidation spike at the desired voltage. Our catalyst still hasn’t come in, and we’re waiting on that to proceed. Because actual work is stalled, we’re working on finding other types of improvements and modifications to the system.

September 3-8, 2011

Author: Niko

The Chemicals still haven’t arrived. We have been assured that they have been send on the 1st and everything we have ordered so far has taken two to three days. However, our chemicals still aren’t here. We are hoping that they arrive tomorrow, as this is the last day that we can work full time on our project.

I spent this week preparing for the fact that, starting Monday, I won’t be in the lab every day and won’t be able to work with everybody every day. This included discussing what other people are up to and making sure that Felix and I can do our experiments without needing other people to help us.

September 5-9, 2011

Author: Felix Chung

Accomplishments:

1. Final week! Dealt with wrap-up and assorted housekeeping tasks.
2. Went to represent iGEM and Biomedical Engineering at the first year engineering fair.

Results

Not much went on this week besides dealing with minutia. Overall, the first year engineering fair was an amazing success as far as PR went - I managed to engage several students about biomedical engineering and its possibilities, with a specific focus on synthetic biology. Aside from that, in the absence of a catalyst for our electrochemical cell, we cleaned up our bench and began discussing the presentation with the rest of our team.