igem 2014: uc santa cruz bioe project

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iGEM 2014: UC-Santa Cruz-BioE Sai Edara - Biomolecular Eng Aaron Maloney - Bioelectronics Eng Marshall Porter - Biomolecular Eng David Dillon - Biomolecular Eng Christian Pettet - Biomolecular Eng Arjun Sandhu - Biomolecular Eng Ansley Tanoto Alex Ng

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Project Summary of the iGEM 2014 team: UC Santa Cruz BioE. A microbial fuel cell (MFC) uses bacteria to break down organic compounds found in waste water and generate an electric current. My colleagues and I intend to genetically engineering the bacteria Shewanella oneidensis in ways that will make it will participate in energy production more efficiently. We will be on of the two teams competing in the international genetically engineered machines (iGEM) competition to represent the UC Santa Cruz Banana Slugs on an international stage! Please consider helping us fund this project and/or giving it exposure. http://tinyurl.com/ne9cqbb

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Page 1: iGEM 2014: UC Santa Cruz BioE Project

iGEM 2014: UC-Santa Cruz-BioE

Sai Edara - Biomolecular EngAaron Maloney - Bioelectronics EngMarshall Porter - Biomolecular Eng

David Dillon - Biomolecular EngChristian Pettet - Biomolecular Eng

Arjun Sandhu - Biomolecular EngAnsley Tanoto

Alex Ng

Page 2: iGEM 2014: UC Santa Cruz BioE Project

● Undergraduate Synthetic Biology Competition put on by MIT.

What is iGEM?

Page 3: iGEM 2014: UC Santa Cruz BioE Project

● Undergraduate Synthetic Biology Competition put on by MIT.● Students from universities around the world work with a kit of biological

parts, and parts they design, to build biological systems

What is iGEM?

Page 4: iGEM 2014: UC Santa Cruz BioE Project

● Undergraduate Synthetic Biology Competition put on by MIT.● Students from universities around the world work with a kit of biological

parts, and parts they design, to build biological systems

What is iGEM?

● UCSC’s debut year at the jamboree held in Boston, MA will be "...the largest single event in the history of the iGEM (International Genetically Engineered Machines) competition and synthetic biology "

Page 5: iGEM 2014: UC Santa Cruz BioE Project

● This year we are working with the bacteria Shewanella oneidensis to increase efficiency of a microbial fuel cell, a technology capable of turning waste water treatment into a power generating process.

iGEM at UC Santa Cruz

Page 6: iGEM 2014: UC Santa Cruz BioE Project

Waste-water treatment ● Treatment of waste water can be divided into three main steps

1. Heavy and light materials are removed by separation in a holding tank2. Microorganisms are used to break down organic matter3. Water is disinfected to be reintroduced to environment

http://en.wikipedia.org/wiki/Sewage_treatment

Page 7: iGEM 2014: UC Santa Cruz BioE Project

The bacteria Shewanella oneidensis

● Can live in both environments with or without oxygen

● Can reduce poisonous heavy metal

● Has “electrogenic” properties allowing it to generate electricity in a Microbial Fuel Cell (MFC).

http://www.newscientist.com/article/dn9526-bacteria-made-to-sprout-conducting-nanowires.html#.U9gp_LEzCM0

Page 8: iGEM 2014: UC Santa Cruz BioE Project

What is an MFC?● Microbes Break down

Carbohydrates● Transfers electrons to anode,

which then flow to the cathode● Protons pass through

permeable membrane● Protons and electrons react

with oxygen to make clean water

● Can be implemented into secondary treatment of waste-water to allow for power generation[5] http://www.sciencebuddies.

org/Files/3665/5/Energy_img033.jpg

Page 9: iGEM 2014: UC Santa Cruz BioE Project

Physical Design● A lot of previous research has

looked at structural design● Two main points

○ Large surface area on electrode

○ Close distance between electrodes

● 3D Model for casing Designed by iGEM 2013 Team Bielefeld (Germany).

● Files converted into 3D printer files and printed. http://2013.igem.org/Team:Bielefeld-

Germany/Project/MFC

Page 10: iGEM 2014: UC Santa Cruz BioE Project

Our Project● We believe the bacteria which drive the power generation of an

MFC can be genetically engineered to create more power● Design MFC with increased efficiency by

○ Altering metabolism of our electrogenic bacteria○ Modifying growth pattern of biofilm formation

● Two pronged approach, each with potential to improve efficiency alone

Page 11: iGEM 2014: UC Santa Cruz BioE Project

Energy Balance and Coulombic Efficiency

● The process of metabolism and electron transfer is complex.

● The cell itself uses up some of the energy in other processes

● One such process is metabolite generation, which reduces coulombic efficiency.[1]

● We plan to redirect metabolism toward a pathway capable of harvesting the lost energy

Page 12: iGEM 2014: UC Santa Cruz BioE Project

● When Shewanella is grown without oxygen, it generates the metabolite acetate from acetyl-coa

Acetate generation

[3]

Page 13: iGEM 2014: UC Santa Cruz BioE Project

● “gate keeper” to the TCA cycle● Converts acetyl-CoA and

Oxaloacetate to Citrate● Diverts Acetyl-CoA from being

converted to Acetate (metabolite)

Citrate Synthase (GltA)

Page 14: iGEM 2014: UC Santa Cruz BioE Project

● Under anaerobic conditions Shewanella is capable of using the oxidative branch of TCA, which will produce more energy lost by metabolite generation

● Use of oxidative branch is reliant on Citrate Synthase activity

Oxidative branch

Oxidative branch of TCA

Page 15: iGEM 2014: UC Santa Cruz BioE Project

Citrate Synthase● Under the anaerobic conditions citrate synthase activity

reduced by over one half due to downregulation of the gltA gene coding for citrate synthase [3]

● In our project we will recover this activity using an expression plasmid

(gene deletion)[3]

Page 16: iGEM 2014: UC Santa Cruz BioE Project

● Magnitude of electron transfer reliant on surface area of the anode○ More surface area allows more bacteria

to transfer electrons○ Growth of bacteria in biofilm allows for a

dense community to grown in one area● Growth of Shewanella in anaerobic

conditions leads of down regulation of biofilm production, and biofilm density is lost

Biofilm

Steps in Biofilm growth: 1 2 3 4 5

http://en.wikipedia.org/wiki/Biofilm

Page 17: iGEM 2014: UC Santa Cruz BioE Project

Biofilm● Biofilm formation in Shewanella is

controlled by the gene mxdA, which regulates levels of c-di-GMP

● Upon deletion of mxdA, biofilm biomass decreases (fig A, mxdA)

● Biomass also decreases when switching from oxic to anoxic growth (fig B, control) but is retained when a gene similar to mxdA is expressed (fig B, VCA0956)[7]

● We hope to express mxdA in anoxic conditions [3] to increase biofilm density

A

B

[7]

Page 18: iGEM 2014: UC Santa Cruz BioE Project

Citations1. Korneel Rabaey, ed. Bioelectrochemical systems: from extracellular electron transfer to biotechnological application. IWA

publishing, 2010.2. Franks, Ashley E., and Kelly P. Nevin. "Microbial fuel cells, a current review." Energies 3.5 (2010): 899-919.3. Brutinel ED, Gralnick JA. Anomalies of the anaerobic tricarboxylic acid cycle in Shewanella oneidensis revealed by Tn-seq.

Mol Microbiol. 2012 Oct;86(2):273-83. doi: 10.1111/j.1365-2958.2012.08196.x. Epub 2012 Aug 27. PubMed PMID: 22925268.

4. Papagianni M. Recent advances in engineering the central carbon metabolism of industrially important bacteria. Microb Cell Fact. 2012 Apr 30;11:50. doi: 10.1186/1475-2859-11-50. Review. PubMed PMID: 22545791; PubMed Central PMCID: PMC3461431

5. Rabaey K, Verstraete W. Microbial fuel cells: novel biotechnology for energy generation. Trends Biotechnol. 2005 Jun;23(6):291-8. Review. PubMed PMID: 15922081.

6. Beliaev, Alex S., et al. "Gene and protein expression profiles of Shewanella oneidensis during anaerobic growth with different electron acceptors." Omics: a journal of integrative biology 6.1 (2002): 39-60.

7. Thormann, Kai M., et al. "Control of formation and cellular detachment from Shewanella oneidensis MR-1 biofilms by cyclic di-GMP." Journal of Bacteriology 188.7 (2006): 2681-2691.