A Synthetic Future: Microcompartments, Nanoparticles

and the BioBattery

Alix Blackshaw, Lisza Bruder, Mackenzie Coatham, Ashley Duncan, Jeffrey Fischer, Fan Mo, Kirsten Rosler, Roxanne Shank, Megan Torry, and Hans-Joachim Wieden

Overview

Our Project: Develop compartments in Bacteria

Two Approaches to Compartmentalization:

nanoparticles and microcompartments

Technological Applications

Human Practices: Science in Southern Alberta

Compartmentalization

TEM image of a plant parenchyma cell.

From “The Cell: A Molecular Approach “4th edition (Cooper and Hausman 2006)

Chloroplast

Nucleus

Vacuole

Golgi ApparatusMitochondria

Eukaryotes segregate metabolic processes into compartments

Prokaryotes lack distinct organelles

Compartmentalization:A Foundational Advance

Co-localizing cellular processes in bacteria will improve the efficiency of the system

Reduce Cross-Talk

Isolate Toxic Components

Concentrate Substances

Bring Components

into close proximity

Isolating Toxic Components through Compartmentalization

EM image of nanoparticles formed in the presence of Mms6.

From Prozorov et al., 2007

pLacI RBS Mms6 dT

Mms6 from Magnetospirillum

magneticum

IPTG inducible construct was

produced in collaboration with the

UNIVERSITY OF ALBERTA

A Self-Assembling Cage

Lumazine synthase from Aquifex aeolicus forms 60 subunit icosahedral capsids

Monomer Homopentamer

Lumazine Synthase Capsid

Targeting Strategy

Glutamate mutations produce a highly negative interior of the

lumazine synthase microcompartment

A positively charged protein may be targeted to the inside

of the microcompartment

Wild-type (-15 formal charge) 4X Glu mutant (-40 formal charge)

BioFusion Vectors

10 Arginine residues (R10) were attached to either the N- or C-terminus of YFP

C- or N- terminal

R10 Fusion Vector

YFP

C- or N- terminal

R10 Fusion Vector

Restriction

Digestion LigationC- or N- terminal

R10 YFP

Express

λexcitation = 514 nm

λemission = 526-527 nm

Fo

ld C

ha

ng

e in

Flu

ore

sc

en

ce

Fluorescence of YFP Constructs

0

5

10

15

20

Volume of interior – 299 - 369 nm3 (Fluorescent proteins are 56 nm3)

Pore size – 1.93 nm

Modelling the Capsid

1.93 nm

diameter

8.3-8.9 nm

diameter

Studying Co-localization through FRET

λemission = 527 nmλexcitation = 439 nm λemission = 476 nm

Distance dependent mechanism

Diameter of microcompartment is 8.9 nm

Characterizing Co-localization –the Mechanism

Pro

tein

co

ncentr

ation

Increasing Concentration of

Ara and IPTG

LS

FP

Characterizing Co-localization –the Mechanism

Pro

tein

concentr

ation

Time

LSFP

No Ara, IPTG induced

Characterizing Co-localization –the Mechanism

Pro

tein

co

ncentr

ation

Time

LS

FP

Induced with IPTG and Ara

Future Applications

•Co-localization of metabolic proteins or processes

•Sequestering toxic gene products

•Delivery capsidAnode Cathode

Electron Flow

O2 + 2H+

H20

Cyanobacteria

CO2 + H20 + light

(CH2O)n + 02 + H20

H+

e-

Mediator

Sunlight

Science in Southern Alberta

Opinion of Genetic

Engineering

Opinion of

Synthetic Biology

Science in Southern Alberta

Interest in Science

Interest in Scientific Career

Accomplishments

•31 new BioBrick parts

•Improved on U of L 2008 iGEM

team’s riboswitch construct

•Collaborated with the University

of Alberta, Valencia and TUDelft

•Created and characterized fusion cloning vectors for targeting proteins into

Lumazine synthase microcompartments, where the proteins remained functional

Acknowledgements

The Wieden Lab

The Kothe Lab

John

Thibault

Dave

Franz

University

of

Lethbridge

Students’

Union

Nathan

Puhl

Sebastian

Machula