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 – slim@ntu.edu.sg

• 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…