Biomod/2011/Harvard/HarvarDNAnos:LabNotebook Nick

Monday (2011-06-06)

 * This morning we began with a brainstorming session and discussed a few different box designs that we hope to represent as caDNAno files.
 * Throughout the day, I designed a six sided box with parallel helices and a hole in one side. My initial inclination is to create a staple-lock-staple mesh over the designed hole to contain cargo that is loaded into the box pre-folding.  This mesh could (irreversibly) be removed using a key strand or a designed restriction enzyme site at staple-staple interfaces.
 * Perkons_Box1.json


 * Below is one of the scariest looking caDNAno screens after hitting auto-staple
 * Below is a screenshot of the cleaner, and much less intimidating, CanDo output. It looks like the design was a success!  Now to design a cover for the hole that can be opened by a separate mechanism...

Tuesday (2011-06-07)

 * This morning I continued to work on my caDNAno file from yesterday, this time attempting to insure that all crossovers occurred after a minimum of 8 nucleotide lengths on any given helix (to insure stability). Given that this was nearly impossible, I decided to start again, this time designing a six-sided box with a hinged lid.
 * Later this morning, we made 2D rectangular DNA origami with materials already available in the Yin lab and attempted to photograph it on the AFM.
 * This afternoon we met with Tom and Wei, postdocs here in the Yin lab at the Wyss Institute. We discussed our initial box ideas and some new ones.  Here is what we learned/spoke about:
 * 1) For a five nanometer gold particle, the interior of any surface we design should have a diameter of at least 20 nm to account for a hydrodynamic shell present about the particle
 * 2) The m13 sequence (which we will be using for our origami) is 7,249 nucleotides long; any design we create in caDNAno should have a scaffold of no more than 7,249 nucleotides
 * 3) The possibility of designing a spherical "box" that could be opened along its equator
 * 4) The possibility of designing a box with a spring mechanism for opening a hinged lid

Wednesday (2011-06-08)

 * This morning I began work on Tom's idea of a spring-loaded box. I used caDNAno to essentially design a five sided box without the lid (I plan to design the lid shortly).  However, the .json file that I have for this five-sided box can be used for either my first idea of being covered by a staple-staple or staple-lock-staple mesh or for Tom's idea of a lid with a spring mechanism.
 * Perkons_OpenBox.json


 * CanDo Output for Open Box Design


 * Later in the afternoon, we devoted a significant portion of time to discussing spherical and rounded designs.

Thursday (2011-06-09)

 * This morning I began work on a frisbee shape and a hemisphere, ideas taken from the following article: 
 * ... I did nothing else this morning because re-designing a hemisphere on caDNAno was more time consuming than I ever could have imagined. So much so that we decided to ask for high-res monitors from the Yin lab to help us out.
 * This afternoon, however, I went to the Yin Lab group meeting, which was interesting! We look forward to presenting our preliminary ideas to the group next Thursday.

Friday (2011-06-10)

 * This morning I returned to the my work on the hemisphere. Luckily it was a success and I was able to recreate the figures contained within the Science article I referenced yesterday.
 * Perkons_Hemisphere.json


 * While my caDNAno file was accurate, CanDo didn't necessarily agree (it actually kind of looks like an infinity symbol).
 * caDNAno File Screenshot


 * CanDo File Screenshot


 * I spent the remainder of today experimenting with Maya, a 3D design software program free to college students!


 * Our Week 1 Team Photo!

Monday (2011-06-13)

 * This morning I began work on a program that specifies whether the staple strand at a point on the sphere we have replicated is either on the interior or exterior face of the sphere. The input of our program is the coordinates of a specific base in terms of phi and theta on the surface of a sphere in our Maya 3D model of the sphere, and the output is the caDNAno location of that base, along with the direction of the staple strand at that location on the helix.




 * My Massive Data Table in Progress!

Tuesday (2011-06-14)

 * Today I continued work on my huge spreadsheet of direction values as Shwinn began writing the code for a Hashmap function in Python that mapped each base value to coordinates on Sherrie's caDNAno file and Evan's model of our sphere.


 * Evan's 3D Model of the Sphere

Wednesday (2011-06-15)

 * Today I finished the Excel file and began to teach myself basic Python and Java coding techniques.
 * We also folded a 2D rectangular origami structure today in the lab!
 * My First Program! (while quite rudimentary)

Thursday (2011-06-16)

 * This morning I did some error checking on the Excel document I completed yesterday. Then, I worked on isolating the staple strands located on the seam of Sherrie's sphere.  My ultimate goal is to manipulate these strands so that they may be cleaved by a restriction enzyme (via a specific palindromic sequence), UV light (via the incorporation of azobenzene), or via DTT (via the incorporation of a disulfide bond).
 * This afternoon we had a group meeting with Professor Yin, Professor Shih, Adam, and several graduate students and postdocs from both labs.
 * We talked about some new ideas for box opening mechanisms and attachment schemes for cargo, including the possibility of using a complete torus shape.


 * Congrats Bruins! (2011 Stanley Cup Champions)

Friday (2011-06-17)

 * This morning I spent a lot of time working on the Project section of our wiki. Check it out at HarvarDNAnos Project Page!
 * I also investigated the activity of the Restriction Enzyme NOT1 and began designing a mechanism of incorporating its recognition sequence into the seam of the sphere in order to facilitate opening of the sphere. In specific, I designed a construct to test whether or not a restriction enzyme motif would be sufficient to open our sphere.  Note in the image below, if the hairpin is open we should be able to detect this on a gel, and this will show that a given amount of pre-cut excess is sufficient to allow a R.E. to cut but sphere to open.
 * Testing NOT1 Restriction Enzyme


 * Our Week 2 Team Photo!

Monday (2011-06-20)

 * Today I spend the majority of the morning experimenting with Illustrator and Photoshop, as the Yin lab graciously purchased each of us a license for CS5. After getting acquainted I was able to produce by first Illustrator image, a depiction of Restriction Enzyme Scheme as a means to opening the sphere.


 * Opening a Sphere Using the R.E. NOT1

Tuesday (2011-06-21)

 * Today we had the opportunity to use a lot of expensive equipment! First we worked with Steve in using the Dynamic Light Scattering device to determine the hydrodynamic radius of gold nanoparticles.  Later in the day, we worked with Adam on the TEM and looked at E.coli cells infected with bacteriophages.


 * What a Typical TEM Looks Like

Wednesday (2011-06-22)

 * Today we worked with Steve again from the Shih lab and were able to synthesize our own colloidal gold particles of various sizes (from about 5nm to 50 nm in diameter)
 * This morning I also began working on preparing solutions of several pH ranges for synthesis of our disulfide linked strands.
 * Whilst doing other things, we also ran a series of gels containing samples from yesterday of our attempt to make a nanoparticle chain. While we had a little difficulty managing the thin acrylamide gel the first time, we were able to get a much cleaner result our second time around.


 * Our First Acrylamide Gel of an Attempt to Make a Nanoparticle Chain

Thursday (2011-06-23)

 * This morning I worked on determining the hydrodynamic radium of five of the species of gold nanoparticles we synthesized yesterday with Peter. Below you will find some data outputs from the DLS machine, which overall showed that the more gold particles from a stock solution of 5nm particles introduced to a given volume of AuCl yielded smaller gold aggregates (more particles = more locations for the aqueous gold to bind, such that an individual pre-formed gold particle will be smaller if there are more pre-formed gold particles in solution.
 * This afternoon I worked on updating the wiki and used Illustrator to create a model for the Disulfide Model of opening our sphere.
 * Later this afternoon we presented our work at the Yin lab group meeting!


 * Disulfide Linkers as a Mechanism to Open the Sphere


 * Hydrodynamic Radius of Particles after Addition of 500 uL of 5nm Au Particle Stock Solution

Friday (2011-06-24)

 * This morning I prepared a conjugation of two 5' thiol strands for our disulfide linker test that we will be preparing next week in two solutions (one of pH 7 and the other of pH 8).
 * This morning I also met with one of our faculty mentors, Peng Yin, to talk about our future plans for the BioMod team.
 * This afternoon I also helped Sherrie and Evan add DNA to gold nanoparticles.


 * Our Week 3 Team Photo!

Monday (2011-06-27)

 * This morning I prepared the disulfide test reaction by mixing the disulfide bonded strands with DTT for an hour. Unfortunately, the image I obtained from the Typhoon was distorted because my DNA ladder spilled over into some of my other wells.
 * We also attempted to isolate Gold nanoparticles with discrete numbers of attached oligos using an agarose gel, but unfortunately we think the gel was too thick and after five hours very little banding had occurred. We plan to re-do this experiment with Steve on Wednesday.
 * Preparing my samples!

Tuesday (2011-06-28)

 * Today I began designing the interior handles for attaching nanoparticles inside the sphere. We also began folding the sphere today!
 * Meanwhile, I also began to retest the disulfide reduction reaction by DTT from yesterday.  The typhoon was constantly in use though, so I plan to image my gel first thing tomorrow morning!

Wednesday (2011-06-29)

 * The first thing I did this morning was image the results of my disulfide reaction on the Typhoon, and it looks promising!
 * This afternoon we also used the AFM to image our sphere after purification through a 2% agarose gel
 * Personally, I modeled the strand displacement mechanism for releasing cargo inside the sphere
 * As a team we also worked with Adam in preparing our sphere samples for imaging on the TEM tomorrow morning with Wei
 * The above image shows Disulfide linkage and subsequent cutting by DTT at pH 7 & 8. The first non-ladder lane (reading left to right) contains disulfide linkage (un-cut) at pH 7, the second lane contains cut disulfide bonds at pH 7, the third lane contains un-cut bonds at pH 8, and the fourth lane contains cut disulfide bonds at pH 8.  The large bands in all four lanes contain unconjugated thiols (or cut disulfide bonds).  In lanes 1 and 3 we can see faint bands, which we believe represent conjugated disulfides.  Our future goal is to beef up the percentage of conjugated disulfides prior to cutting by DTT by finding an optimal pH or solvent mixture.

Thursday (2011-06-30)

 * This morning we worked with Wei on the TEM and imaged our spheres. Unfortunately, we were unable to get a quality image of a folded sphere because of some problems with our staining technique and an incorrect concentration of buffer used in our folding reaction.
 * This afternoon after talking with Peng and William for the Biomod team meeting, we decided to drop the idea of using disulfide linkers in our sphere because of the ability of gold nanoparticles to cleave dithiol bonds, which in our case would facilitate the formation of our disulfide bonds.
 * TEM image of potential sphere, but likely only a water droplet

Friday (2011-07-01)

 * This morning I made an agarose gel again with the following specifications: 1 mL 1.2 M MgCl2, 8 uL Sybr gold, 120 mL .5x TBE, 2.4g agarose to purify our prepared sphere samples
 * Following this, I again made AuNP with Steve and used the DLS to determine the hydrodynamic radius of my synthesized particles
 * Afterwards I began the protocol for conjugating AuNP to DNA by adding phosphene to the mixture of my synthesized Gold Nanoparticles
 * [[Media:PrePhosphene Original.pdf]]

Monday (2011-07-04)

 * Happy Fourth of July from Boston!

Tuesday (2011-07-05)

 * This morning I continued the conjugation protocol for AuNP and DNA by centrifuging my mixture from last Friday after adding salt, methanol, and phosphene buffer
 * Afterwards I measured samples from the various stages of this protocol for AuNP aggregation
 * We also began re-folding the sphere in the proper buffer conditions
 * [[Media:PrePhosphene 4DaysLater.pdf]]
 * [[Media:PostPhosphene PostMixing.pdf]]
 * [[Media:PostSaltAddition.pdf]]
 * The above files are links to the DLS measurements of my samples from various times. The first document, "PrePhosphene_4DaysLater" shows that there was a degree of aggregation of AuNP after sitting at 4 degrees C for four days without the addition of phosphene.  The second measurement shows that the addition of phosphene stabilized the particles and prevented aggregation.  The third measurement file, "PostSaltAddition" shows minimal aggregation of AuNP, but this is probably a result of using the centrifuge (which is required in the procedure).

Wednesday (2011-07-06)

 * This morning we spent a lot of time working on the wiki
 * In the afternoon we used both the AFM and TEM to image our recently re-folded sphere, we have some awesome images that show our open spheres on both TEM and AFM
 * This afternoon I also developed a program, SphereCADBasic, which helps users determine whether a staple is pointing in, out, up, or down at a specified location on the sphere modeled in Han's paper. The program can use either caDNAno coordinates, coordinates from Han's paper, or Theta/Phi coordinates from our Maya3D image, which is in development.
 * SphereCADBasic


 * TEM Image of our unpurified closed spheres


 * TEM Image of our unpurified open spheres


 * Using SphereCADBasic
 * 1) Download SphereCADBasic and open with Microsoft Excel or a compatible spreadsheet viewer (e.g. OpenOffice, NOTE: GoogleDocs cannot open this file)
 * 2) Use any of the three available input options:  caDNAno coordinates, Supplementary Info Coordinates, or Maya 3D Coordinates
 * [[Media:Sphere match.json | caDNAno Sphere File]]
 * Han's Supplementary Info, see page S142
 * Maya File Coming Soon!

Thursday (2011-07-07)

 * This morning I worked with Evan in preparing TEM samples of our boxes, spheres and gold nanoparticles in preparation for attaching U1C sequence to gold nanoparticles
 * Later this afternoon we worked with Adam on the AFM so that we could examine purified open spheres
 * This afternoon we also attended the Yin Group Meeting

Friday (2011-07-08)

 * This morning Evan and I prepared our attachment of Au to the U1C DNA Sequence
 * We also attempted to use the TEM to image our samples prepared yesterday but accidentally disengaged the vacuum and were no longer able to use the machine for the day

Monday (2011-07-11)

 * This morning I spent a lot of time working on getting some kinks out of SphereCADv.2, please check it out and send me feedback. The .zip file contains all necessary files to run the caDNAno file and a .txt file containing instructions on how to run the program.
 * We also began the process of gel purification of our AuNP conjugated to DNA strands, a protocol that we hope to continue on Wednesday with Steve.

 


 * Nickstestpage