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Abstract
Acknowledgements
Currently no tools are available to the synthe8c biology community that allow for the separa8on of engineered biochemical processes in order to improve characteriza8on and the stability of these systems. To achieve this will development microcompartments that will mimic the compartmentaliza8on which occurs in eukaryotes, including the ability to selec8vely target proteins into this compartment. The system we are crea8ng is based on an engineered lumazine synthase protein containing point muta8ons to generate a highly nega8ve interior. This protein can a?ract highly posi8ve 10 arginines (R10) tags, enabling a selec8ve targe8ng system. We have successfully created the cloning vectors to introduce these signal pep8des to either the N‐ or C‐terminus of any protein of interest. We have modeled the lumazine synthase microcompartment and shown that it has a volume between 299 and 369 nm3 which is able to accommodate proteins of up 80 kDa ins size. The pore size however will not allow for the escape of these proteins from the compartment. In order to test co‐localisa8on of two proteins into the microcompartemnt we will test this system using the fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. An alterna8ve approach to compartmentaliza8on is the crea8on of nanopar8cles which we are going to op8mize using the nanopar8cle producing protein Mms6 within Escherichia coli system.
A Synthe)c Future: Microcompartments, Nanopar)cles and the BioBa;ery
References
Alix Blackshaw, Lisza Bruder, Mackenzie Coatham, Ashley Duncan, Jeffrey Fischer, Fan Mo, Kirsten Rosler, Roxanne Shank, Megan Torry, and Hans‐Joachim Wieden
University of Lethbridge, 4401 University Drive Lethbridge, Alberta, Canada T1K 3M4
Nanopar8cles
Thanks to David Franz, Nathan Puhl, John Thibault, Sebas8an Machula, Tony Russell, Bill Hamman and the University of Alberta iGEM 2009 team.
Thanks to the University of Lethbridge Department of Chemistry and Biochemistry and the Faculty of Arts and Science.
Prozorov et al. (2007). Protein‐mediated synthesis of uniform superparamagne8c magne8te nanocrystals. Adv. Funct. Mater. 17, 951‐957
Seebeck et al. (2006). A simple tagging system for protein encapula8on. J. Am. Chem. Soc. 128, 4516‐4517
Tsujimura et al. (2001). Photosynthe8c bioelectrochemical cell u8lizing cyanobacteria and water‐genera8ng oxidase. Enzyme and Microbial Technology. 29, 225‐231
Microcompartments
Parts Submi?ed to the Registry
Lumazine synthase (LS) from Aquifex aeolicus was mutated at four posi8ons by introducing glutamates. The system was modeled and the muta8ons were found to result in a more nega8ve formal charge of ‐480 in comparison to the wild type ‐180 for a 60 subunit capsid.
Figure 1. Electrosta8c surface poten8al map of two subunits of the pentamer mutant LS of the a) inside face and b) outside face. Areas of posi8ve and nega8ve charge are shown in blue and red, respec8vely.
a) b)
Figure 3. A representa8on of cyan and yellow fluorescent proteins (FP) within the LS microcompartment.
a) b)
Figure 2. Electrosta8c surface poten8al map of 11 subunits map with 1 subunit cap removed of the a) wild type and b) mutant LS. Areas of posi8ve and nega8ve charge are shown in blue and red, respec8vely.
Figure 5. Fluorescence of cell extracts containing R10 YFP constructs.
Figure 7. The final LS microcompartment co‐localiza8on construct.
R 10 Tail
pLacI
RBS Lum
azine
Syntha
se
dT pB
ad
RBS
YFP
R 10 Tail
dT
RBS
CFP
dT Tet
R
Inverter
Figure 6. Schema8c representa8on of FRET occurring between the donor CFP (excita8on 439 nm, emission 476 nm) and acceptor YFP (excita8on 514 nm, emission 527 nm).
The nanopar8cle producing protein (Mms6) has been constructed based on the protein found in Magnetospirillum magneBcum.
Figure 8. The final Mms6 construct.
pLacI RBS Mms6 dT
Figure 9. Electron microscope images of magne8te nanopar8cles obtained by co‐precipita8on of FeCl2 and FeCl3 in solu8on a) without protein and b) with Mms6 (Prozorov et al., 2007).
a) b)
Future Direc8ons
Complete the assembly of the lumazine synthase microcompartment co‐localiza8on construct.
Characterize the lumazine synthase capsids Size of assembled capsids
Characterize the method of co‐localiza8on Independently control lumazine synthase and fluorescent protein expression
Localize photosynthe8c protein(s) within the lumazine synthase microcompartment
Characteriza8on and op8miza8on nanopar8cle produc8on by localiza8on of the Mms6 protein.
Assembly and op8miza8on of the photosynthe8c fuel cell.
C‐Terminus R10 Tail
BBa_K249005
C‐Terminus R10 – dT
BBa_S04261
Microcompartment:
Nanopar8cle:
Improved Previous Parts:
pBAD – TetR Inverter – mRBS – R10 YFP – dT
BBa_K249017
pBAD – TetR Inverter – mRBS –YFP R10 – dT
BBa_K249014
Lumazine Synthase
BBa_K249002
N‐Terminus R10 Tail
BBa_K249004
EYFP – Fusion Standard
BBa_K249006
Fusion Standard – YFP
BBa_K249008 pBad – TetR inverter
BBa_K249001
Lumazine Synthase – dT
BBa_S04259
RBS – N‐Terminus R10 Tail
BBa_S04262
EYFP – R10 Tail – dT
BBa_S04264
mRBS – R10 FP
BBa_S04266
Mms6
BBa_K249016
pLacI –RBS – Mms6 ‐ dT
BBa_K249019
Mms6 – dT
BBa_S04268
Riboswitch
BBa_K249026
pTetR – RBS – YFP
BBa_K249000
pStrong – Riboswitch – cheZ – dT
BBa_K249028
GFP – dT
BBa_S04263
pStrong – Riboswitch – cheZ
BBa_S04270
cheZ – dT
BBa_S04271
Riboswitch – cheZ – dT
BBa_S04272
Riboswitch – GFP – dT
BBa_S04270
Judging Criteria
Further modeling demonstrated that the capsid interior diameter is between 83 and 89 nm. The fluorescent protein (FP) volume was calculated to be approximately 55.49 nm3 showing that between 5 or 6 proteins can fit into the capsid unable to exit through the 1.93 nm pores.
Fold Change in Fluorescence
Buffer
DH5α
C‐R 10 YFP
C‐R 10 YFP
+ Ara
N‐R 10 YFP
+ Ara
N‐R 10 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
Figure 4. Schema8c representa8on of the construc8on strategy of R10 fusion proteins (Biofusion, Silver Lab) which can be targeted to the LS microcompartment by the electrosta8c forces.
Wavelength (nm)
Excita8on of YFP
Intensity
Emission of CFP
Had a fun summer, now we are here!!! Complete and submit the iGEM 2009 Judging form Create and share a descrip8on of the team’s project Present a Poster and Talk at the iGEM Jamboree Enter informa8on detailing new standard BioBrick Parts DNA submi?ed for new BioBrick Parts Demonstrate new BioBrick Parts of own design and device Characterize or improve an exis8ng BioBrick Part or Device Help another iGEM team Detail a new approach to an issue of Human Prac8ce Answered the four iGEM safety ques8ons
Had a fun summer, now we are here!!! Complete and submit the iGEM 2009 Judging form Create and share a descrip8on of the team’s project Present a Poster and Talk at the iGEM Jamboree Enter informa8on detailing new standard BioBrick Parts DNA submi?ed for new BioBrick Parts Demonstrate func8on new BioBrick Parts Characterize or improve an exis8ng BioBrick Part or Device Help another iGEM team Detail a new approach to an issue of Human Prac8ce Answered the four iGEM safety ques8ons