Meeting #3 (4/29/12)
The link to the presentation can be found here.
- Idea: Improve H2 production in bacteria using RNA and MAGE
- MAGE: Accelerated evolution - only done with E. coli so far
- During replication, the replication fragments allow for the oligonucleotide to hybridize (certain chance per cycle)
- Non-perfect hybridization - non-perfect, local edit
- Goal: Produce bacteria that have been selected to produce high amounts of H2 - link to reporter or antibiotic?
- Scaffold only 2 strands, but do you want to do MAGE (getting better over cycles) versus huge library of mutations
- Edits in different spots - otherwise the mutations will effectively undo each other (non-incremental)
- Large Scale (kb) - insertion into genome
- Small Edits - library
- How to select for those that produce hydrogen?
- RNA scaffold only in E. coli as well
- Idea: Co-evolve phages with bacteria using MAGE
- Goal: Try to evolve phage at a rate comparable to the bacterial evolution in hospitals
- PACE: Phage Assisted Continuous Evolution - use phage with another plasmid - grow in E. coli with a part of the E. coli so phage and "E. coli need to coexist
- Fluid flow "thing"
- PACE is more difficult to get to work
- Idea: Use MAGE to engineer microbiome
- Can't use MAGE - need to evolve through E. coli
- But if microbiome is engineered - people are trying it but is beyond the scope of a summer
- Idea: What happens to bacteria which are constantly exposed to H2-accelerating scaffolds?
- Bacteria doesn't like H2 scaffold - lots of pipetting?
- Bacteria wants to kick out scaffold - if keep reintroducing, then will be exposed for a longer period of time but maybe some long-term changes?
- May evolve to even with scaffold - no hydrogen but not known for sure
- Idea: Improve speed and mobility of modified RNAP motors
- How to move cargo over a decent distance?
- Limits of distance?
- Intermediates versus one step
- DNA Walker - DNA machine - which walks on the 100 bp limit
- RNAP - no 100bp limit anymore - can use PCR to walk on something as long as you want
- GUGUGUC to fall off - can halt using idea from before
- Idea: Create longer tracks for RNAP motors using existing stop signals
- Goal: Custom make a track
- Start with template - product as long as template - attach start and stop
- Unless template is de novo - no template to start with
- Adam and Mingjie are doing this!
- Idea: Bind DNA to microtubules to allow in vivo movement
- Adam-approved =D
- Mammalian cells - microtubules have protein already attached - snaptag
- Can covalent bond to a particular chemical - will auto bind to microtubules
- Ralf is trying this
- Can microtubule stretch the DNA?
- Would be cool of polymerases could walk on microtubules - would then achieve DNA streching - to stretch DNA in a straight configuration
- Important for everything including sequencing,
- Challenging but not impossible
- Stretching from making of microtubule
- Difficulty: bind while stretch - stretch = strong entropic force
- DNA stretch by origami - then remove - stretch across microtubule
- Origami binds to two different parts of microtubule
- Point? but interesting
- Build origami - native length longer - stretch while folding
- Idea: How viable are DNA origami structures inside the body?
- Nucleases will attack
- Immune reactions
- Problems only solved in preliminary stages
- Modify structures to optimize for stability - chemically
- Idea: Logic gating using DNA origami
- Circuit-based DNA computing
- Strand displacement - use circuits - with things bumping around
- Can use to open/close DNA origami?
- Would be a fun project - very design - immediately visible effects
- Idea: Cascade of DNA boxes
- Consider size of magnets and field strengths
- Folding needs Mg positive ions to prevent the repulsion of the negatively charged DNA
- Idea: Penetrate cells using aptamers/cell-penetrating enzymes
- Peptide called RGD
- Sort of done - but not really - acknowledged to be a good idea
- Cy3 is a little fluorescent dye
- Cancer cell with folate receptors
- Aptamers go to the cell surface - can signal endocytosis?
- Combine with changes of DNA origami
- Would also be quite fun
- Idea: Making RNA versions of DNA origami structures
- Would be cool
- Why did people use DNA instead of RNA - key reason RNA structures are harder to predict - less strict structuring
- Idea: Measure half-life of nano-structure degradation
- Same way to screen for good folding would lead to screening for degradation as well
- Idea: Incorporate DNA into genome and make RNA origami out of transcribed RNA
- Not defined shape like rectangle
- Can't arrange functional elements in those ways
- But can do other thigns like attach other RNA which binds to GFP or attach 2 hydrogen producing enzymes that want to be near each other
- Either in vitro or in vivo
- Mingjie approves!
- Start with E. coli which will eliminate the second challenge
- Can easily stop from being translated - no ribosome binding site - but then work around degradaton
- Look at why rRNA and why they dont' get degraded - different mechanism or form unqiue structure that isnt' recognized
- CAN WE MAKE A MACHINE OUT OF rRNA?
- Church lab studying specific parts of rRNA
- Can get the DNA in
- Same problem of how to get it to fold - self assembly
- Miscellaneous comments
- Ways to figure out of something folded properly - fluorescence
- Allows for parallel screening
- Polymerase halt - can without the compliment and it would effectively "stop" unless it adds the wrong thing which it doesn't like to do
Meeting #2 (4/29/12)
- A Logic-Gated Nanorobot for Targeted Transport of Molecular Payload 
- Organization of Intracellular Reactions with Rationally Designed RNA Assemblies 
- Synthetic Biology Moving into the Clinic 
- A Tightly Regulated Molecular Motor Based upon T7 RNA Polymerase 
- Programming cells by multiplex genome engineering and accelerated evolution 
- Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome 
- Other applications of box structure using things like particles and logic gates? Continue last year’s project? Make a cascade?
- Let’s look at problems to solve, not necessarily applications of ideas
- In Young
- Use MAGE to create novel bacterial lines
- Apply MAGE to photosensing - capturing light to create energy?
- Using bacteria to make even higher-density fuels that are therefore more economical
- Seems like the MAGE process would be a lot of work
- Using walkers seem interesting. Can we make them faster? We should make a DNA walker that can actually walk along microtubules. We know DNA can bind to microtubules. Interesting since they aren’t so sure ATP can be used.
- It seems like the most successful things have been done by taking something from the cell and using it rather than trying to create something.
- Train of DNA polymerases with Nyanocat passengers.
- Interesting how the genomes we work with are too small.
- Using a bigger genome? It seems like it would be useful.
- Novel geometries attached at center forming sphere
- Increasing sensitivity of boxes (sphere of boxes more effective than single)
- DNA scaffolds inside cells
- nanostructures (spikes that puncture cells) - insert DNA scaffolds or complementary-binding mRNA strands which can inhibit free-floating mRNA strands already found in the cell which are needed specifically by cancer cells and not by normal cells.
- Is inserting into cell issue?
- Scaffold to accelerate photosynthesis - accelerate assembly line (flow of electrons - could we accelerate this flow using DNA scaffold? treat it like a conductor?
- Phage/Virus with special RNA, the reverse transcriptase is used to create DNA, self-assembles inside cell. Synthetic life used to alter other cells?
- Using DNA nanotech in multicellular organisms or even just eukaryotes
- Using magnetic field to activate or deactivate nanorobot? Maybe the magnetic field can be used to not just activate/deactivate but also to localize the DNA nanorobots in select places like cancer tumors. Use several magnets that spin around to create an undulating force which can make the nanorobots have rotary-like motions
- Using magnetics so that two particular units only bind to each other and create an activated unit.
- How long do DNA boxes last outside of the human body?
- Testing how the geometry affects how this origami structure lasts in the body?
- Tailored restriction enzymes to cut up an existing nanorobot?
- One box releases restriction enzymes that cuts something else
- In Young
- Structures of varying stability
- Need to amplify
- Alter the microbiome so that weight-loss drugs produced in the gut can be artificially produced, production induced by food intake.
- Weight loss is associated with stress-inducers or chemicals produced in times of dietary stress that thus accelerate fat burning (and the related system of proportional response). Can we make bacteria that produce these stress-response chemicals based on how much food the human eats?
- In Young
- Why not use MAGE to accelerate the evolution of fat-loss inducing organisms found in the microbiome
- Use dna sequences to stop polymerase in its tracks
- Making bacteria that can better design dna scaffolds
- Use fluorescently labelled NTPS to create a new kind of sequencing
- Using two part of gfp that are split apart and attached to two ends of the dna scaffold and fluoresce when they snap together
- Using MAGE to accelerate the evolution of the human microbiome over a long period of time, what happens if you reinsert it into the human once you have a dna scaffold, put it into the cell, dunno what happens afterward? put that into a mage and see what happens, will the cells change? maybe hydrogen production increases? once you put the through evolution continuously, optimize that scaffold? selectively alter that scaffold, choose one with highest H2 production
- Venter paper → synthetic cell. what happens when you put synthetic life into a machine?
- Objection: not evolution on entire genome, need to choose specific parts
- In Young
- Economic objection to constantly putting in this scaffold
- More sustainable in-vivo: construction by transforming the origami sequence into the genome and using reporter genes for analysis
- Insert into genome, have cell transcribe the part that is the scaffold
- Simpler concepts that can actually be achieved over a summer
- Something with multiple stages of achievement in case we don’t achieve the entire goal realistically
- Mechanism of logic gates
- Develop logic gates → build a computer, encoder, already have AND gate, whole system, OR, XOR, more binding sites? what NOT means for biology
- General Topics:
- MAGE (various applications) - Ian Choi
- DNA Walker (increase speed) - In Young
- DNA Origami (geometry, stability, restriction enzymes, limits) - Wes
- DNA Delivery (magnetic targeting, logic gates) - Val
- Modifying cell DNA (phage, scaffold, microbiome) Jacob
- Proof of concepts of In Vivo Ideas - Gina :)
- 10 Bad ideas 10 Not so Good Ideas
- Creating the Veritas symbol out of DNA origami
- Train!!!! with passengers!!
- Fiddling with microbiome of living organisms to tailor synthesize specific compounds upon introduction of particles (like food)
- Better controlling DNA walkers by choosing start and end points (with specific DNA sequences)
- DNA minecraft / world!!
- More logistical details
- CadNano tutorials
Meeting #1 (4/25/12)
Challenges and opportunities for structural DNA nanotechnology 
Dynamic DNA nanotechnology using strand-displacement reactions 
Harnessing biological motors to engineer systems for nanoscale transport and assembly 
Brainstorm (bolded ideas spurred positive discussion about feasiblity):
Competition is open to any ideas at molecular level
Should be DNA related in the end for the presentation
DNA/RNA can be much more useful than cells/proteins
Anything done with simple proteins can be very huge though.
People use DNA not because it’s the best but because it was just what was available
Decide what exactly we want to do by mid or late May. Meet every week?
Meet this time next week
Cascade of boxes
In vivo is potentially feasible
Use logic and/or gates to self-assemble at high accuracy
How did you decide on last year’s project?
Box on chalkboard
Use DNA to hook proteins together?
Assembly within a cell?
Nanoparticle assembly with help of DNA?
Pop up assembly?
Plate gridded DNA with nanoparticles? Semiconducting? Model after graphene?
Use the accelerated high-throughput evolution to create bacteria that can make DNA for you.
Use mages - use sixty-base DNA and put into cells.
Replaces some of the okazaki fragments
Use an interior red flag with fluorescent tag that shows when assembly failed?
Separate bad ones with filter?
Magnetic nanobeads? Use them? Or use them to speed up assembly?
UV crosslinking as a new method for DNA origami?
We want them to assemble complementary though
Make buckyballs out of DNA?
Use DNA AND RNA together?
ATPase + DNA = robot?
How do you make it?
What does it do?
Use DNA nanostructures for sequencing? Somehow?
Nanopores/protein pores for sequencing?
Attach magnet to DNA nanocargos and use a super magnet above patient to localize only around tumors
What is the crucial capability that we can add?
Mingjie - not our field's advantage
Use DNA boxes to eat bacteria
Make perpetual loop cascade
Problem of error
Need to add stuff to keep powering... use ATP?
Magnetic tracks for DNA robots?
Use cloud computing to screen for dynamics of crosstalk
PRINT DNA structures
Good for making libraries
DNA walkers - what’s the application?
Can we use enzymes?
The nice thing is that they are programmable
But the main drawback is that they are very, very slow
Can we speed it up?
Can we use heat?
We are talking about often is DNA replication
Rate limiting step is often the biased random walk
Temperature-mediated walk based on how similar the DNA strands are
Problem is that it isn’t sustainable
Artificial immune system
Can we do stuff in the cell?
How do we measure the success of our final outcome?
Concretely: Can we see technically correct outcomes?
How do we know it actually worked?
Imaging neurons in a zebrafish - any applications?
Some scientists made a computer that would play tic tac toe with RNA
Interfering with the mRNA found in cancer cells
How do DNA computers work?
Using stochastic motion to power DNA nanorobots
Next time, come with five or six big ideas
Come up with four good ideas
What do you think is the most difficult part?
Then come up with ten really bad ideas and explain!
Can we read a few more reviews because they really help catalyze ideas?
Shawn’s nanorobot paper
Meet next week and present