FYP @ BeANs Lab Info SessionFYP @ BeANs Lab Info Session 2012 – 2013 The BeANs Lab Bioengineered...
Transcript of FYP @ BeANs Lab Info SessionFYP @ BeANs Lab Info Session 2012 – 2013 The BeANs Lab Bioengineered...
FYP @ BeANs Lab Info Session
2012 – 2013
The BeANs Lab Bioengineered and Applied Nanomaterials Laboratory
Logistics • Lab: N1.3-B3-15
• The mentors – 1 Postdoc – 3 Graduate Students
• The supervisor – Asst. Prof. Sierin Lim – N1.3-B3-06 – +65 +514 1085 – [email protected]
• Website: www.ntu.edu.sg/home/slim
Projects
Future Healthcare • Drug delivery
system • MRI contrast
agent
Sustainable Earth • Photosynthetic
Electrochemical Cell (PEC)
PROTEIN
The protein cages & chlorophasts
Image: Dalmau & Lim, et al., Biotechnology & Bioengineering,
vol. 104(4), 654-664 (2008)
Image: Kichoefer et al., PNAS, vol.102(12), 4348-52 (2005)
Image: Johnson et al., Structure, vol. 13, 637–648 (2005)
Image: Wikipedia
Chloroplast 2-10 x 1 µm
Vault 40 x 70 nm 96 subunits
E2 25 nm
60 subunits
Ferritin 15 nm
24 subunits
Project Organizations
DNA Molecular cloning (PCR, mutagenesis)
Expression / Production Fermentation
(Growth condition optimization)
Purification FPLC
(IEX, HIC, SEC, ultrafiltration)
Characterization Structure
(TEM, DLS, CD) Activity
(Colorimetric assay)
Modification Functionalization (ligand attachment)
Further Analysis Sequence comparison
(data mining, phylogenetic tree)
Future Healthcare Screening
High efficiency photosystem
Isolation
Prototype Design
Immobilization Current measurement
Sustainable Earth
BIE/12/091: Characterization of Ferritin-bound Anticancer Drug
• Ferritin – Iron storage protein – Converts metallic (Fe/Mn) salt to
magnetic nanoparticle that is a potential MRI contrast agent.
– Nonspecific encapsulation of limited number of drug molecules is possible in vitro
• Objectives: – Optimize specific binding of
both iron and metal-based anticancer drugs (theranostic)
– Determine the relaxivity values of the theranostic agent
Simsek et al. J Magn. Magnetic Mater. 293: 509 (2005) Sana et al. Biointerphases 5:FA48-FA52 (2010) http://pubs.rsc.org/en/Content/ArticleLanding/2012/CC/c1cc15189d http://www.springerlink.com/content/l5njh78275656160/?MUD=MP
AfFtn AfFtn-AA CDDP-N
BIE/12/092: Determining Cellular Targeting of EGFR-Functionalized Ferritin-bound Anticancer Drug
• Ferritin – Iron storage protein – Converts metallic (Fe/Mn) salt to
magnetic nanoparticle that is a potential MRI contrast agent.
– Nonspecific encapsulation of limited number of drug molecules is possible in vitro
• Objectives: – Optimize binding of targeting
ligand to ferritin – Determine binding efficiency
of the targeted ferritin to EGFR-expressing cancer cells
Simsek et al. J Magn. Magnetic Mater. 293: 509 (2005) Sana et al. Biointerphases 5:FA48-FA52 (2010) http://pubs.rsc.org/en/Content/ArticleLanding/2012/CC/c1cc15189d http://www.springerlink.com/content/l5njh78275656160/?MUD=MP
AfFtn AfFtn-AA
BIE/12/093: Determining Cellular Activity of (Mn)Ferritin-bound Anticancer Drug
• Ferritin – Iron storage protein – Converts metallic (Fe/Mn) salt to
magnetic nanoparticle that is a potential MRI contrast agent.
– Nonspecific encapsulation of limited number of drug molecules is possible in vitro
• Objectives: – Develop cell-line handling
protocol – Determine pharmacokinetic
parameters on breast and liver cancer cell-lines
Simsek et al. J Magn. Magnetic Mater. 293: 509 (2005) Sana et al. Biointerphases 5:FA48-FA52 (2010) http://pubs.rsc.org/en/Content/ArticleLanding/2012/CC/c1cc15189d http://www.springerlink.com/content/l5njh78275656160/?MUD=MP
AfFtn AfFtn-AA CDDP-N
BIE/12/094: Magnetic Characterization of Protein-cage-templated Nanoparticle
• E2 protein cage: Part of the pyruvate dehydrogenase
multienzyme complex Can be engineered to carry non-native
functionality, e.g.: Drug carrier Metal carrier
• Objectives: – Optimize iron-loading protocol – Characterize the nanoparticle
synthesized within the cage • Magnetic using vibrating sample
magnetometry • Size using dynamic light scattering
Tosha T et al. (2008) PNAS 105(47):18182 http://dx.doi.org/10.1002/bit.21988 http://dx.doi.org/ 10.1002/adfm.201200052
E2- LE6 + Fe2+
E2- LFer + Fe2+
E2- LE6
• E2-LE6: ~1800 Fe/cage • E2-LFer: ~2000 Fe/cage
BIE/12/095: Synthesis of Metal Nanoparticle Using E2 Protein as Template
• E2 protein cage: Part of the pyruvate dehydrogenase
multienzyme complex Can be engineered to carry non-native
functionality, e.g.: Drug carrier Metal carrier
• Objectives: – Screen binding affinity of other
metal types – Characterize the nanoparticle
synthesized within the cage • Elemental analysis using XPS
Tosha T et al. (2008) PNAS 105(47):18182 http://dx.doi.org/10.1002/bit.21988 http://dx.doi.org/ 10.1002/adfm.201200052
E2- LE6 + Fe2+
E2- LFer + Fe2+
E2- LE6
• E2-LE6: ~1800 Fe/cage • E2-LFer: ~2000 Fe/cage
• Vault – natural nanocapsule for drug delivery – Composed of major vault protein (MVP, >70%), vault poly
(ADP-ribose) polymerase (VPARP) - Includes minimal interaction domain (mINT domain), telomerase-associated protein 1 (TEP1), untranslated RNA
– MVP (blue) domain 3 to 5 interacts specifically with mINT (golden). The region is referred to as iMVP.
– Controlling the dissociation of mINT has an implication in controlling the release of therapeutic agents.
BIE/12/096: Process Development for the Production of Engineered Natriuretic Peptide
• Objectives: – Produce working plasmid for the
production of CD-NP-INT – Develop process for the
production of the engineered natriuretic peptide
Kickhoefer, V.A., et al., PNAS,
2005. 102(12): p. 4348-4352.
http://www.physiology.dal.ca/people/faculty/pubs/48_348.pdf http://www.jbc.org/content/283/50/35003.full
• Objectives: – Develop co-culture condition
for the PEC – Examine the open-circuit
voltage and current produced
• Photosynthesis – Electron production based on water
splitting reaction in the presence of light.
– Cyanobacteria can be used to generate carbon-neutral electricity in a photosynthetic electrochemical cell (PEC)
BIE/12/097: Examining Mutualistic Symbiosis of E. coli and Cyanobacteria
Co-culture in a Photosynthetic Electrochemical Cell
Alberts, B., et al., Molecular Cell Biology of the Cell (4th Edition). 2002: Garland Science Lam, K.B., et al., Sensors and Actuators, B: Chemical, 2006. 117(2): p. 480-487.
E. coli: - Uses O2 - Produces CO2
Cyanobacteria: - Uses CO2 - Produces O2
• Objectives: – Integrate carbon-nanotube
(CNT) on carbon-cloth electrode
– Examine the voltage/current improvement
• Photosynthesis – Electron production based on water
splitting reaction in the presence of light.
– Cyanobacteria can be used to generate carbon-neutral electricity in a photosynthetic electrochemical cell (PEC)
BIE/12/097: Modification of anode electrode in cyanobacteria
photosynthetic electrochemical cell
Alberts, B., et al., Molecular Cell Biology of the Cell (4th Edition). 2002: Garland Science Lam, K.B., et al., Sensors and Actuators, B: Chemical, 2006. 117(2): p. 480-487.
The Expectations
Hours • 10 hours/week in the lab (minimum) • 2-3 hours reading
Log book
• Maintain daily journal • Include objective, all protocols, results, and conclusions
Update • Weekly • Attendance at group meetings; usually on Fridays
Present • Once per semester • 30-40 min
Expand • All project objectives are expandable • Differentiate between B+, A-, and A
Data • Publishable = A+
The Next Steps
• Email your top 3 choices for pre-allocation
• And wait…