Microcompartments, Nanoparticles and the BioBattery Alix Blackshaw, - - PowerPoint PPT Presentation

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 Apparatus Mitochondria

Eukaryotes segregate metabolic processes into compartments Prokaryotes lack distinct organelles

Compartmentalization: A Foundational Advance

Co-localizing cellular processes in bacteria will improve the efficiency

• f 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

• f 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 Ligation C- or N- terminal R10 YFP Express

λexcitation = 514 nm λemission = 526-527 nm Fold Change in Fluorescence

Fluorescence of YFP Constructs

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 nm λexcitation = 439 nm λemission = 476 nm

Distance dependent mechanism Diameter of microcompartment is 8.9 nm

Characterizing Co-localization – the Mechanism

Protein concentration Increasing Concentration of Ara and IPTG

LS FP

Characterizing Co-localization – the Mechanism

Protein concentration Time

LS FP

No Ara, IPTG induced

Characterizing Co-localization – the Mechanism

Protein concentration Time

LS FP

Induced with IPTG and Ara

Future Applications

• Co-localization of

metabolic proteins or processes

• Sequestering toxic

gene products

• Delivery capsid

Anode 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
• f 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

• f

Lethbridge Students’ Union

Nathan Puhl Sebastian Machula