Harvard iGEM 2006

Our Vision: modular drug delivery 1. 2. cell A cell B Editing slide - make three slides, bigger, fix blue blobs so they look like cells and that idea is more clear Good afternoon and welcome. On behalf of the 2006 Harvard iGEM team I’d like to speak to you about our research this summer. We began the summer with a few goals, and I will discuss the first. Inspired by Dr. William Shih’s work with DNA nanotechnology at Harvard medical school, we decided it would be very useful to develop a container to package and carry new drugs. We decided to attack this problem from two angles - one group worked on development of a novel box-like DNA structure to protect proteins while another group worked on cell-surface targeting. cell C targeting packaging

Why DNA? Strong Watson-Crick base pairing Covalent modifications Relatively inexpensive Self-assembles Highly programmable Why might we be interested in using DNA to build three dimensional structures? Well, as it turns out, there are a number of properties of DNA that make it desirable to work with. Unlike protein engineering, construction with DNA is surprisingly predictable. It relies entirely on standard Watson-Crick base pairing which is very strong. Furthermore it’s very easy to make modifications to DNA with things like restriction enzymes and other covalent modifiers. Synthesis of short strands of DNA is inexpesive. And most importantly, DNA is highly programmable. Because we know that its structure will be based on Watson-Crick base pairing, we can design the DNA we order to fold up in certain ways with simple heating and cooling.

How: engineered crossing over How exactly do we stick this DNA together?

How: scaffolded origami N. Seeman P. Rothemund W. Shih et. al

Our Design: a novel structure courtesy Shawn Douglas

Our Design: a novel structure courtesy Shawn Douglas

Evidence: EM images 15.5 nm

Evidence: EM images 27.6 nm 30.5 nm

Evidence: protection assay Protected biotin on the inside of our container from large streptavidin beads Streptavidin bead Streptavidin bead Payload protected Control: biotin bound

Harvard iGEM 2006

Adaptamers Thrombin aptamer Streptavidin aptamer Linking region We can contain, now how to d we deliver. “two strategies” Change color of whiteness Add text to define “aptamer” and “adaptamer” Give it structure by discussing what we’re trying to do Aptamer: nucleic acid sequence that can bind a substrate with high specificity and affinity.

Why develop adaptamers? To offer a new way to target substrates to cells. To create a tool for studying cell-cell interactions. To provide a foundation for engineered catalysts. happy colorful things. Highlight key words.

Adaptamer testing Bead surface Adaptamer + thrombin streptavidin adaptamer Adaptamer + thrombin thrombin anti-thrombin antibody (-) control: naked beads Change thrombin color. Remove A15., move text towards image. Bead surface

Adaptamer quenching + + Displacement strand change color of displacement strand, stop blinding pp (yellow). Remove stray line on top left. Talk about “machine”ness of displacement, is a feature of aptamer. Change grey background of bar graph. Plus sign cut off on the bottom +

Streptavidin on the cell surface Binds strongly to biotin molecule Used to bind biotinylated nucleic acids or peptides Big idea: Streptavidin protein expressed on the cell surface can be used to target any biotinylated DNA/protein to the cell surface. McDevitt, 1999 Identify image of streptavidin. Bring out the big idea immediately.

Lpp-OmpA surface display J. Francisco C. Earhart G. Georgiou 1992 Surface protein extracellular Lpp (lipoprotein) signal peptide outer membrane periplasm OmpA (outer membrane protein A) transmembrane domains

BioBricks assembly of protein domains I. Phillips P. Silver 2006 5’…TCTAGAG XbaI TACTAGT…3’ SpeI Standard BioBrick 5’…TCTAGA_ XbaI _ACTAGT…3’ SpeI Protein domain BioBrick TCTAGT Mixed 6-bp mixed site Reading frame maintained Confusing coloring of green parts/promoter, and purple mixed/coding sequences. Promoter RBS Lpp OmpA Streptavidin Lac

Expression of fusion protein in frame Anti-Streptavidin Anti-Histidine Inducible expression Histidine tag at end, so construct in frame Streptavidin domain recognized by Ab Explain the identifications. Put the conclusion as title or actual text. Maybe cut down to one lane, one construct.

Harvard iGEM 2006

From cyanobacteria... ...to E. coli Photosynthetic Circadian rhythm Evolved over billions of years ...to E. coli Model organism for synthetic biology BioBrick registry

Applications of a Bio-oscillator 12PM 12PM Clock Nightlight Timed drug delivery Pharmaceutical processes Bio-circuitry Investigate natural systems A+B->C A+E->F 6PM 6AM 6PM output http://www.cellzome.com/img/pathways.jpg Circuitry D+C->E D+F->P 12AM 12AM Time 22

Cyanobacteria Bio-oscillator The Repressilator Cyanobacteria Bio-oscillator Lac λ-cI Tet Elowitz et al. 2000 ` Integral to existing electronic circuits. 46.1 Useful for future complex, time-dependent biocircuitry. Existing synthetic oscillators not that useful: Fluorescence 10h Time 24h 23

The Kai Clock in Cyanobacteria KaiC autophosphorylates and dephosphorylates KaiA promotes phosphorylation KaiB inhibits KaiA Transcription-translation independent Period: 14-60 h A B P P C P P b Phosphorylation of KaiC P P output Time

Achievements Goal: reconstitute the cyanobacteria Kai oscillator in E. coli Created KaiA, KaiB, and KaiC BioBricks. Combined the above with registry parts to form functional BioBricks. Expressed Kai proteins in E. coli and verified interaction.

Results: Constructs Created We’ve made the following constructs: Kai genes Lac promoter + Kai genes KaiA + KaiC and KaiB + KaiC (with promoters)

Results: Proteins Interact in E. coli Western blot image Constructs transformed in E. coli Cultures sampled at OD 0.5 Western blot probed with anti-KaiC antibodies

Results: Proteins Interact in E. coli Western blot image A A C B C C

Results: Proteins Interact in E. coli Western blot image A A C B C C

Results: Proteins Interact in E. coli Western blot image A A C B C C

Results: Proteins Interact in E. coli Western blot image Conclusion: KaiA and KaiC are expressed and interacting KaiB not verified, but results consistent with predictions

Further challenges A A B B C C Verify oscillation in E. coli. P Synchronization problem Between cells Within cell Solution: pulsed expression Time long version

Harvard iGEM 2006

Acknowledgements Advisors Teaching Fellows George Church Pamela Silver William Shih Radhika Nagpal Jagesh Shah Alain Viel Teaching Fellows Nicholas Stroustrup Shawn Douglas Chris Doucette