Team:FHS Frederick MD

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	Team FHS_Frederick_MD

Official Team Profile

We are interested in creating a microbial fuel cell that utilizes anaerobic bacteria to produce electricity. In order to optimize the growth conditions in the fuel cell, a fluorescent protein marker will be added so to visualize bacterial growth. We plan to implement an oxygen-sensitive promoter to induce expression of the glowing gene. This should ensure that bacteria only grow under anaerobic conditions. This would lead to the creation of a genetic construct that can be deposited back into the “toolbox” parts repository for iGEM.

Goals

Microbial Fuel Cells

A microbial fuel cell is a device that converts the chemical reactions of bacteria into electricity. Within the fuel cell certain bacteria under anaerobic conditions will remove the electrons from organic matter and transfer them to an anode, which will then transfer the electrons through a circuit to a cathode. The current and voltage produced by this process is what creates the electricity required to power certain objects such as a light bulb.

The bacteria that we are currently creating is meant to optimize the microbial fuel cell's potential, as bacteria that will glow under anaerobic conditions will reveal any weaknesses in the fuel cell, structural or otherwise, which can then be assessed and dealt with.

Gene Design

(Kyle and Jonathon, briefly summarize how NirB and LOV work together.)

NirB Promoter

The NirB gene is reliant on a fumarate and nitrate reductase (FNR), which allows the promoter to activate when there is no oxygen present, as well as facilitates in the regulation of transcription that is responsible for the growth under anaerobic conditions. When oxygen is not present, the 4Fe-4S complex helps join the FNR components. As this happens, it becomes a protein that attaches to the NirB part of the DNA, which results in the production of the LOV gene.

In an experiment by Morales, sterile oil was glossed over the tube containing the bacteria. The bacteria were then able to use the oxygen until the levels were completely depleted, resulting in a fully anaerobic environment. Once fully anaerobic, the bacteria continued to produce the LOV gene product.

Morales, Pedro Luis Dorado. "pNirB + Gene encoding ZsGreen1." . http://parts.igem.org/Part:BBa_K763002 (accessed June 16, 2014).

LOV Domain

(Jonathon, this is your area to describe how we engineered the LOV gene.)

See LOV Domain for a more detailed description of how we constructed the gene.

LOV stands for light oxygen voltage. It is a sensor protein that detects the presence of blue light(365nm). In its wild type form it is used by higher plants, fungi, and bacteria. In higher plants LOV controls phototropism and chloroplasts relocation. In this form it absorbs blue light(365nm) and in the wild state flavin mononuclotides(FMN) link to cysteine. This results in LOV not being able to emit green light(495nm) due to FMN. We choose to modify LOV as are anaerobic environment and growth indicator. We choose love over green fluorescent protiens(GFP) due to the fact that GFPs are completely depend on molecular oxygen to glow. However due to FMN LOV can not release green light(495nm). Thomas Drepper found a solution to this problem Drepper and his fellow researchers realized the effects of FMN and found away to remove it. By eliminating the cysteine amino acid FMN had nothing to bind to. Following Dreppers model we removed the cysteine amino acid from bacillus subtilis.

To create the LOV strand, we initially created cultures of E.coli containing the plasmid pBB1MCS2. We chose this plasmid due to its ability to transform a wide range of bacteria, including our desired host bacterial strain of Schwenella odeneidensis for the LOV gene. We grew it in kanamycin-rich plates to eliminate all possible bacterial colonies lacking pBB1MCS2. Using the Quia prep spin mini prepkit, we extracted the plasmid from the E.coli bacteria. We tested the purity of the uncut plasmid through electrophoresis.

We then grew cultures of Bacillus subtilis that contained the desired LOV domian. Extraction of the genomic DNA from B.subtilis was accomplished through a boil prep procedure.

We then digested LOV and NirB into two separate preps containing the plasmid pB1C3 already treated with EcoRI and PstI,which originated from the iGEM 3A assembly kit.

Ligation of the two digests of NirB and LOV were completed fusing them to the plasmid pB1C3. Using these new plasmids, we then commenced on transformation of the LyoComp cells. However the transformation efficiency was very low.. After two failed transformations, we decided to create our own chemically competent cells,thus created E.coli NE1U beta cells. We then performed another digestion, as well as ligation, with LOV and NirB. We completed another transformation using E.coli NE1U Beta bacteria with the prepared plasmids. Following the incubation period, we saw growth on all plates cultured,with the exception of the negative control. Thus we can conclude that we extracted the two plasmids using the mini prep kit and successfully transformed those plasmids into the bacteria to show expression.

Methods

(This is Dillon's domain.)

3A Assembly

We used the 3A, or 3 antibody, assembly kit in order to transform E.coli with two genes, the LOV gene and the NirB gene. These genes will allow for further work with Schwenella bacteria in the anaerobic microbial fuel cell. We then used the mini-prep components of the kit to purify our plasmid. We verified the plasmid's presence through electrophoresis and further sequence analysis.

Notebook

The following link will direct you to the ArsBiotechnica website which houses all the protocols we followed through the last year to reach our goals of purifying the plasmid the was transformed into our E.coli bacteria

http://arsbiotechnica.org/w/Procedures

[http://arsbiotechnica.org/w/Article:002-001 20 Feb 2014]: Received iGEM supplies
[http://arsbiotechnica.org/w/Record:002-010 03 Apr 2014]: Digested NirB and LOV inserts
[http://arsbiotechnica.org/w/Record:002-011 10 Apr 2014]: Ligated NirB and LOV genes into pSB1C3
[http://arsbiotechnica.org/w/Record:002-012 14 Apr 2014]: Transformed E. coli with LOV and NirB constructs
[http://arsbiotechnica.org/w/Record:002-013 21 Apr 2014]: Ligated NirB and LOV genes into pSB1C3
[http://arsbiotechnica.org/w/Record:002-014 28 Apr 2014]: Transformed E. coli with LOV and NirB constructs
[http://arsbiotechnica.org/w/Record:002-015 01 May 2014]: Prepared TSS buffer
[http://arsbiotechnica.org/w/Record:002-016 05 May 2014]: Ligatied the RFP plasmid
[http://arsbiotechnica.org/w/Record:002-017 08 May 2014]: Subcultured E. coli NEB10 Beta
[http://arsbiotechnica.org/w/Record:002-018 08 May 2014]: Tested transformation efficiency of competent cells
[http://arsbiotechnica.org/w/Record:002-020 12 May 2014]: Digested NirB, LOV, pSB1C3 DNA fragments with EcoRI and PstI
[http://arsbiotechnica.org/w/Record:002-021 15 May 2014]: Ligated NirB and LOV into pSB1C3
[http://arsbiotechnica.org/w/Record:002-022 20 May 2014]: Transformed E. coli with LOV and NirB constructs
[http://arsbiotechnica.org/w/Record:002-023 29 May 2014]: Cultured individual NirB and LOV colonies
[http://arsbiotechnica.org/w/Record:002-024 02 Jun 2014]: Purified NirB and LOV plasmids
[http://arsbiotechnica.org/w/Record:002-025 03 Jun 2014]: Visualized NirB and LOV plasmids on a gel
[http://arsbiotechnica.org/w/Record:002-026 04 Jun 2014]: Recultured NirB and LOV transformants
[http://arsbiotechnica.org/w/Record:002-027 05 Jun 2014]: Extracted plasmid DNA from bacterial transformants
[http://arsbiotechnica.org/w/Record:002-028 06 Jun 2014]: Observed plasmid DNA on a gel
[http://arsbiotechnica.org/w/Record:002-029 09 Jun 2014]: Submitted samples for sequencing
[http://arsbiotechnica.org/w/Record:002-030 11 Jun 2014]: Resubmitted DNA for sequencing

Results/Conclusions

What did you achieve over the course of your semester?

Safety

What safety precautions did your team take? Did you take a safety training course? Were you supervised at all times in the lab?

We wore gloves and lab coats during each lab experiment in order to maintain sterility Goggles were worn during lab in order to protect our eyes Supervision was a necessity. Either Mr. Trice or Dr. Rozak were always present during all lab experiments.

Attributions

This is our team of four students and three mentors. Everyone has contributed equally to this project both in and out of the lab.

	Kyle Andrushko is an Eagle scout on the road to becoming an optometrist. Having recently graduated from Frederick High school, Kyele is planning on attending UMBC for his undergraduate degree with a major in biology and a minor in business. John's Hopkins medical school will be his next step after taking the OAT. Kyele will be participating in the iGEMs competition in Boston this summer. He has just finished up his first year of iGEMs.
	Dillon Kestner is a Frederick High School graduate who has been inspired by the iGEMs club as well as his mentors Mark Trice and Dave Rozak to pursue a career in neuroscience. He plans to attend a local community college in order to continue being involved in his high school's iGEMs club and mentor future participants before transferring to Muhlenberg College to acquire a doctorate in neuroscience. Dillon looks ahead to the trip to Boston and seeing his group's work pay off as well as all the work put in by groups from all over the world.
	Jonathon Soward is an 18 year-old who loves the study of life. Jonathon attributes his love of STEM to a string of inspiring STEM teachers at his school, including Mark Trice, Shelley Miller, and Joyce Tuten. His teachers, as well as his diligence and work ethic, has enabled him to decide upon pursuing a degree in both dentistry and microbiology. Jonathon has been a member of the iGEM team at Frederick High School for the past two years. Jonathon's nickname is "master pipettor" because of his amazing pipetting skills!
	Alan Nguyen is a recent graduate of Frederick High School and incoming Freshman to Mount St. Mary's University. Alan has taken an interest in biology and its many forms. This interest has given him the drive to pursue a career in medicine, a choice that he wouldn't have made without his AP Biology teacher, Mr. Trice's inspiration. A two year veteran of the iGEM group, Alan is excited to see the fruits of his group's labor as it unfolds at the iGEMS competition held at MIT.
	Mark Trice is a teacher at Frederick High School, where he teachers classes such as Chemistry, Physics, Biology, and Advanced Placement Biology. He also serves as the vice president on the board for the nonprofit organization, Ars Biotechnica. Mark has worked with his iGEM club for two years and is so excited to see this project come to fruition.
	David Rozak is a research scientist at the United States Army Medical Research Institute for Infectious Diseases and a founding director of the nonprofit organization, Ars Biotechnica, Inc. Dave has been working with the Frederick High School Bioengineering Club for two years and served as an advisor to our iGEM team.
	Gary Lopez ...

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Human Practices

As renewable energy and alternative energy resources have become more and more important in the world we live in today, answers, not suggestions, must be found. The research we enacted will allow naturally-occurring bacteria in the soil to generate electricity, rather than traditional methods, such as coal. Through the genetic engineering of the bacteria, we will be able to enhance the bacteria's ability to deposit electrons on the electrodes to produce electricity.

Renewable Energy

(Kyle: How do fuel cell help produce clean renewable energy?) The renewable energy being produced by the fuel cell is the electricity. With the growing of energy scarcity this produces a clean renewable source of energy with readily available use of nutrients.

Clean Water

The bacteria within microbial fuel cells will eat away at many forms of organic matter found in wastewater, thereby fulfilling the role of a treatment plant, and creating a clean water source.

There are also no waste products produced by microbial fuel cells, as the energy they generate is taken from the electrons of the organic matter they digest, therefore making pollutants such as nitrogen and other toxic byproducts of no concern.

Basic Research

Researchers could use our fluorescent gene to determine the length of time it takes from initial placement of the bacteria in the soil until the time electrical energy is generated. Research could be done to amplify the gene and cause the bacteria to generate more energy in a more timely time frame.

Researchers could also use the gene to determine the optimal bacterial concentration that needs to be implemented in order to maximize energy output.

With the glowing gene, researchers could also look at optimal soil types i.e. what nutrients are in the soil, the moisture amount, the soil analysis, etc. Certain soils may generate more electricity or more nutrients may need to be added to soil in order to maximize performance. Farmers may also be able to look at what crops produce certain nutrients and then plan from there as to how to generate electricity.

The gene could also be examined in order to increase electrical output on a larger scale and thus increasing fuel cell performance.

Public Awareness

(Jonathon: Discuss how our meeting with Cardin helped raise public awareness for STEM.)

This year the Frederick High iGEMs team is not only on the fast track to Boston but also to recognition. Purely through word of mouth Maryland senator Ben Cardin came to Frederick High.

The team presented their research on biofuel cells from the previous years and explained in detail why they won their division at last years science fair and what they were currently working on.

Thanks to Cardin's interest in out budding iGEM team, we received some local news coverage. Given this opportunity the team members took the time to also highlight the importance of STEM, expressing how STEM programs such as AP Biology, Chemistry, Environmental Science, and Calculus were a great chance at getting their feet wet and explore STEM.

This media coverage provoked the local Career and Technology center at Frederick Community College to try making their own iGEMs teams with the aid of Frederick High's non profit organization created by Mark Trice and Dave Rozak.

The Frederick News Posts covered Senator Cardin's time with the iGEMs group.

http://www.fredericknewspost.com/news/education/education_topics/learning/genetic-research-sparks-science-careers-at-frederick-high/article_a221dc2d-6208-58b9-b45f-7aed61b37ce3.html

'Can we include the picture of the newspaper, or the selfie with Cardin in this section? == Headline text ==