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

● Undergraduate Synthetic Biology Competition put on by MIT.

What is iGEM?

● 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?

● 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 "

● 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

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

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

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

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

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

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

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

Acetate generation

[3]

● “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)

● 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

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]

● 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

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]

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.