So Happy Together

A Cooperative Relationship between Cyanobacteria and Escherichia Coli for the production of biofuels

University of Nevada, Reno - iGEM 2011

iGEM Team Nevada 2011

20 Undergraduates in Biochemisty, Biology, Biotechnology and

Engineering

With Advisers

Dr. Howard Dr. Shintani Dr. Ellison

Introducing

Concept from Crisis

• Petroleum is a disappearing resource

• As resources diminish, costs increase

PresenterPresentation NotesWe live in a time where we depend on a finite fuel source, alternative forms of fuel must be developed-images from below seal and then going clockwise 1.) country’s size is collated to the amount of oil the own, imbalance or resources requires most of the globe to depended on a very limited resource 2.) graph showing prediction/decrease in oil production 3.) graph showing correlation between hydrocarbon use and global warming 4.) graph depicting the exponential rise in oil cost

Biofuels are the Solution

PresenterPresentation NotesOutline of benefits of biofuel - greenhouse gas emissions decrease with the use of biofuel, biofuels take advantage of the carbon cycle, graph showing that biofuel production, export and consumption is on the rise (this means biofuels are a realistic solution to the fuel crisis for U.S.)

But at What Cost?

• Ethanol increases demand for corn which increase corn prices • Ethanol profits shrink due to an increase in production cost • Fewer crops are planted and farmland prices increase • Large biomass requirements for production in corn

PresenterPresentation NotesWhile biofuels are the solution, food based ethanol production creates agricultural issues food vs. fuel debate

Developing an Alternative Biofuel

• While we have manufactured several different types of biofuels, the cost in comparison to petroleum makes it an expensive alternative • An inexpensive and self sustaining biofuel needs to be developed

PresenterPresentation NotesResearchers have explored E. coli bacteria and alge as producers of biodiesel. However, there is a high cost of growth media and low production levels, still make gasoline a cheaper fuel. In order to make a responsible biofuel we need to overcome all of these obstacles

• E. Coli has the potential to produce large quantities of fatty acids but 30-40% of its production cost goes into supplying growth media How do we reduce this cost? • Cyanobacteria has the unique ability to sustain itself without the cost requirements of E. coli

• Cyanobacteria produces less than half the amount of biofuel produced by E. coli How do we solve these problems?

Developing an Alternative Biofuel

PresenterPresentation NotesResearchers have explored E. coli bacteria and alge as producers of biodiesel. However, there is a high cost of growth media and low production levels, still make gasoline a cheaper fuel. In order to make a responsible biofuel we need to overcome all of these obstacles

Our Project

Cyanobacteria E. coli

Electron micrograph of Synechocystis sp 6803

Cyanobacteria under the microscope

Cyanobacteria

• Cyanobacteria are a phylum of bacteria that obtain their energy through photosynthesis

• Cyanobacteria Synechocystis PCC 6803 is an excellent research model, because its entire genome has been sequenced and it is easily transformed

• Team Utah 2010 graciously supplied us with the cyanobacteria tool kit that allowed us to develop new operons

PresenterPresentation NotesCyanobacteria are a phylum of bacteria that obtain their energy through photosynthesis. The third prokaryote and first photosynthetic organism to have its entire genome sequenced it’s an ideal model for producing glucose in a controlled enviroment. * Talk to team about other points as to why cyano is a good choice, this would be the best time to present this argument *

Developing an Auxotroph

Glycogen

• ADP glucose pyrophosphorylase (AGP)

• Invertase (INV)

• Glucose Facilitator Transporter (GLF)

PresenterPresentation NotesCyanobacteria use photosynthesis to provide energy and carbon skeletons for anabolic processes. During the day excess fixed carbon can be converted to the polysaccharide, glycogen and stored for later use. To engineer Synechocystis to overproduce hexose sugars, we will divert carbon away from the glycogen biosynthetic pathway and towards hexose sugar production. To achieve this goal we will create a null mutation in the gene encoding ADP glucose pyrophosphorylase (AGP). AGP specifically converts glucose to ADP-glucose which is the monomeric precursor to glycogen. Synechocystis AGP knockout mutants have been reported to no longer produce glycogen, but instead accumulate high levels of sucrose. sucrose can easily be converted to the hexose sugars, glucose and fructose, in a reaction catalyzed by the enzyme invertase (INV). Therefore, we will introduce and overexpress the INV gene in the AGP mutant background. To integrate the GLF gene into the Synechocystis genome, we have decided to use an alternative insertion site. In this case we will insert the GLF overexpression gene cassette into the coding region of the thiamin monophosphate pyrophosphorylase (ThiE) gene. The rationale for creating a ThiE knockout mutant is that it will lead to the creation of an auxotophic mutant that will only survive when the grown in the presence of thiamin (Vitamin B1). Therefore, the transgenic Synechocystis will not be able to survive outside laboratory and the chances of environmental contamination will be decreased.

Invertase Development

• ADP glucose pyrophosphorylase (AGP) is a naturally occurring gene in Cyanobacteria that converts ADP-glucose to glycogen

• petBD a promoter that has strong expression in log and stationary phase

• Inverstase (INV) converts ADP-glucose into glucose and fructose

Total Cyanobacteria Genome

Glucose Facilitative Transporter Development

• Thiamine monophosphate pyrophosphorylase (ThiE) a naturally occurring gene in Cyanobacteria that produces thiamine (Vitamin B1)

• Glucose Facilitator Transporter (GLF) a transporter gene that moves glucose and fructose outside of the cell

Total Cyanobacteria Genome

Thiamine monophosphate pyrophosphrylase (ThiE)

Synechocystis Accomplishments

• Chloramphenical resistance cassette needed to be debugged before amplification

pSB1C3 was used as a template for amplification of the coding region for Chloramphenical resistance

• All Synechocystis construct parts are amplified and ready for

Gibson

Our Project

Synechocystis E. coli

Ethanol Production

• Pyruvate Decarboxylase (PDC) converts pyruvate to acetaldehyde (Zymomonas mobilis)

• Alcohol Dehydrogenase (ADH) converts acetaldehyde into ethanol

• σ 70 promoter Constitutive promoter that is not affected by glucose

Ethanol Production

• Pyruvate Decarboxylase Assay

• Alcohol Dehydrogenase Assay

Our Project

Synechocystis E. coli

Fatty Acid Production

• Acyl-ACP is intercepted by Bay Laurel Thioesterase (Umbellularia californica)

• Bay Laurel Thioesterase will turn Acyl-Co A in to C12 and C14 fatty acid derivatives.

Acyl-ACP

Fatty Acid Production

Colorimetric assay results of free fatty acid production versus negative controls

Fatty Acid Production

• Gas chromatography results of Bay Laurel Thioesterase controlled by the σ70 constitutive promoter

Co-Cultivation

So Happy Together!

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10-ß E. coli cells grown in BG-11 with glucose or BG-11, glucose, and NH4Cl.

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BG-11 + 50 mM glucose

BG-11 + 50 mM glucose + 1.0mg/mL NH4Cl

• Confirmed auxotrophies of E. coli

Co-Cultivation: Media

Co-Cultivation: Media

• No significant drop in effectiveness of glucose at 2.5 mM

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Average growth (n=3) of Iq cells grown in BG-11, 0.20% casaminos, and decreasing concentrations of glucose

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BG-11 + .20% casaminos

BG-11 + .20% casaminos + 10 mMglucose

BG-11 + .20% casaminos + 2.5 mMglucose

BG-11 + .20% casaminos + 1.0 mMglucose

Co-Cultivation: Apparatus

• Apparatus must run for several days at a time

• Stop contamination from

external environment

• Prevent cross-contamination as E. coli

travels through dialysis tubing

Co-Cultivation: Apparatus

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Testing of cross-contamination between chambers (test done in BG-11 + 0.2%

casamino acids + 50 mM glucose)

UninoculatedChamber

E. coliChamber

• Solved sequencing errors in pSB1C3 • Apparatus used to co-cultivate different forms of bacteria • Developed an ethanol generator • Developed a medium chain fatty acid generator

Contributions to iGEM

• Production of high value compounds in engineered E. coli

• Self sustaining possibilities for any fermentation system

Future Applications

Human Practices

Sam and Megan presenting at the Rotary Club

Marguerite teaching children about synthetic biology

Student performance at UNR iGEM Concert •Thank you Elaine

Submitted 8 parts to registry Demonstrated functionality of two parts Developed apparatus to co-culture E. coli and

Synechocystis Collaborated with Utah State Collaborated with MIT in debugging pSB1C3

• Thank you Austin Che and the Knight Lab at MIT Developed a method for creating auxotrophies in

Synechocystis for environmental control.

Checklist for Commemoration

Thank You

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