So Happy Together A Cooperative Relationship between Cyanobacteria - - PowerPoint PPT Presentation

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

Biofuels are the Solution

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

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

• 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

Our Project

Cyanobacteria

• E. coli

Electron micrograph of Synechocystis sp 6803

Cyanobacteria under the microscope

Cyanobacteria

• Cyanobacteria are a phylum of bacteria that
• btain 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

Developing an Auxotroph

Glycogen

• ADP glucose pyrophosphorylase (AGP)
• Invertase (INV)
• Glucose Facilitator Transporter (GLF)

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

• utside 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!

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 5 10 15 20 25 30

OD (600 nm) Time (hours) 10-ß E. coli cells grown in BG-11 with glucose or BG-11, glucose, and NH4Cl.

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

• Confirmed auxotrophies of E. coli

Co-Cultivation: Media

Co-Cultivation: Media

• No significant drop in effectiveness of glucose at 2.5 mM

0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 5 10 15 20 25 30

OD (600 nm) Time (hours) Average growth (n=3) of Iq cells grown in BG-11, 0.20% casaminos, and decreasing concentrations of glucose

LB BG-11 BG-11 + .20% casaminos BG-11 + .20% casaminos + 10 mM glucose BG-11 + .20% casaminos + 2.5 mM glucose BG-11 + .20% casaminos + 1.0 mM glucose

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

0.2 0.4 0.6 0.8 1 1.2 21 29 41 48

OD (600 nm) Time (hours) Testing of cross-contamination between chambers (test done in BG-11 + 0.2% casamino acids + 50 mM glucose) Uninoculated Chamber

• E. coli

Chamber

• 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