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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
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