Team:Virtus-Parva Mexico/Project

From 2014.igem.org



The Bio-NEMS drill

The Next Generation in Molecular Machinery





What is it that we do?

The Bio-NEMS drill is the project that we, a group of nanotechnologists have

been working on for almost a year. What started as an idea, became a big part

of our everyday lives and we have done our best to nurture it and watch it grown.

We have prepared different aspects of the project through hard work and it is

our hope that our legacy may continue.



Project Overview

Virtus-Parva is a team of Nanotechnology students who aim to create a better world through love,sympathy and endearment… and through the design and development of a novel technology based on micrometric “drills” that can penetrate diverse organisms.

First and foremost, we studied different procedures in order to synthesize the strongest and smallest nanoparticles we could make. Magnetite is a molecule that derives from iron, and has observable magnetic properties. Because we wanted to put together magnetite and DNA, we had to make them compatible,thrpugh deifferent chemical procedures to allow the binding of this DNa into the particle. At the same time, our biology team was busy extracting, purifying and transforming E. coli DNA to work with. We achieved coiling DNA to nanoparticles. The “Magnetic-DNA” complex we created mimics the nucleosome in DNA supercoiling process. This allows us to have a DNA “chromatin-like” with a magnetic core.

Combining magnetite, and DNA, we are building magnetic-core machines, which can be controlled through external electrical impulses. Because of the shape of our system, it is possible for it to have linear movement depending on the frequency applied to it, which has several potential applications. The main one was to make it a resonator which was our module one. Yet, the project did not stop there. We built a DNA carrier, therefore we started a process to enhance

As we were transforming our E. coli cells, we noticed it wasn’t as fast and efficient as we had hoped, which is how we came up with module two of the project. Quite simply, we wanted to take advantage of the shape of our system and its mobility thanks to magnetism in order to make a more efficient transformation. We were able to verify our method was more efficient by making cells express GFP and RFP, which can then be quantified with optic instruments.




The Idea

The basis for these “drills” will be NEMS, nano electro-mechanical systems, technology.
We took a survey to fellow iGEMers and external people in order to find out how many people knew about the existence of NEMS and if they knew how they worked. Turns out only 36% of survey takers had heard of the term before and of those, only 28% knew what it was!

Given these statistics, it became part of our project to teach newer generations about our subject.

NEMS

NEMS are nanometric electromechanical systems. In this case we take as basis the structure of a resonator which are engineered to make a conversión between energy, such as electric, magnetic, or vibrational into mechanical response.

How exactly do NEMS come into play in our project?

Well, by combining an inorganically synthesized nanoparticle, called magnetite and DNA into what we call BioNEMS drill.

DNA Coiling

Naturally, and as part of the process of transcription DNA is wrapped around protein molecules called histones. The combined loop of DNA and protein is called nucleosome, and this nucleosome is going to be packed into a thread. Our objective was to insert magnetite into this system, in order to convert it into a NEMS and be able to control DNA’s movement using an external stimuli.





The Making

The most important part of our project was the time that we dedicated to seeing results in our lab. Admittedly, it was tough, for we had to do everything in our own time and we had only occasional help from our instructors, for the most part we did everything on our own. Despite all of the size of the challenge, our team rose up to the occasion and in the end all those hours we dedicated in the lab were worth it. Here you can see a short description of what all the things we did in our lab, for a more detailed description check our notebook section.

Inorganic Section

The first part of the synthesis of our magnetite was trying out different methods and characterizing them, to note which method had given us the smallest size nanoparticles. Our first method was synthesis by coprecipitation, of which we prepared nine samples with different concentrations of iron(II) chloride and ammonium hydroxide; from this method we consistently obtained nanoparticles rounding 0.9 to 1nm. Our following method was very similar, but included water in the synthesis: the size of our particles would vary greatly, from 3.89 micrometers to 171 nanometers in size.

After choosing the best method possible, it was time to silanize our magnetite in order for it to be biocompatible with DNA and be able to tie them together. In order for the silanization to take place, we used a solution of TEOS (tetraethoxysilane) dispersed in a medium of water and propanol and dripped this mix slowly onto our magnetite. Just like when we synthesized our particles, we tested different concentrations of TEOS and magnetite, as well as different addition rates in order to observe which combination would give us the smallest possible nanoparticles.

Our results were then characterized by DLS (dynamic light scattering), for which we observed a peak at 39 nm, once coated with TEOS, the peak was moved toward 60 and 80 nm. We also ran our two samples in the IR, comparing the spectra of the pure magnetite and silanized magnetite, we were able to distinguish a peak at 990.2 cm-1 corresponding to a Si-O bond, confirming the correct silanization of the magnetite.



Biological Section

Having done this, we then prepped our protein by resuspending it in a mix of Tris/acetate and EDTA in order to be able to combine it with our DNA. Then we dispersed our magnetite in anhydric toluene and we added the resuspended protein and we added as well some glutaraldehyde as a coupling agent. We also made some batches without any glutaraldehyde, to compare the comparable strength of their bonds.

We then needed to grow and then extract from E. Coli the DNA we were to use for the rest of our project. We tested two different methods: one of which was the well-known mini prep and the other was very similar but without using an enzyme. Using the DNA we have extracted, we then needed to transform these cells to make competent cells.

These cells we had transformed, we then had to purify, by precipitating in presence of ethanol and centrifugation to eliminate supernatant. We did different dilutions of DNA and combined them with our protein, HU. These as well were divided again in gluteraldehyde and no gluteraldehyde and subsecuently in DNA and not DNA. We did some UV characterization for all samples. We discovered that the solutions with glutaraldehyde had stronger bonds between DNA and HU. We prepared some samples: one with glutaraldehyde and DNA-Hu, another just with glutaraldehyde and DNA, the third one just with DNA-HU and the last one just with DNA and prepared them with nanoparticles and ran them through UV.





The Math behind

The first approach to the project in order for it to become feasible was the mathematical model: the number that told us how right or wrong our ideas were. We invite you to visit our modelling page, and find out a lot more about the Bio-NEMS drill and its behaviour. Click on the picture to learn more about this.

Retrieved from "http://2014.igem.org/Team:Virtus-Parva_Mexico/Project"