Difference between revisions of "Team:Cooper Union/Loomino Description"

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<p>There were many alternatives to create fluid flow such as using a system of syringes, or having a gas pressure microfluidic device. The advantage of having a syringe system is that the mechanism needed to pump is relatively simple. By rotating a threaded rod with a motor, we could create linear motion that would compress a syringe, thereby pumping fluids. The issue is that it lacks the ability to pump air, and thus we would encounter large amounts of waste. Also, a primary concern is accuracy. As the stepper motors are able to move within a 1.8 degree rotation excluding half-steps, we are able to more finely control flow rates. A microfluidic device requires more equipment either for lithography or molding PDMS. A gas pressure system would also need to be implemented, and therefore we determined these to be not feasible. </p>
 
<p>There were many alternatives to create fluid flow such as using a system of syringes, or having a gas pressure microfluidic device. The advantage of having a syringe system is that the mechanism needed to pump is relatively simple. By rotating a threaded rod with a motor, we could create linear motion that would compress a syringe, thereby pumping fluids. The issue is that it lacks the ability to pump air, and thus we would encounter large amounts of waste. Also, a primary concern is accuracy. As the stepper motors are able to move within a 1.8 degree rotation excluding half-steps, we are able to more finely control flow rates. A microfluidic device requires more equipment either for lithography or molding PDMS. A gas pressure system would also need to be implemented, and therefore we determined these to be not feasible. </p>
  
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<h2>Thermoelectric Cycling</h2>
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<h2>Thermal Cycling</h2>
 
<p>De Novo Synthesis requires the removal of a heat-labile group at 95 degrees C. The reaction chamber must then be cooled in preparation for the next reagent for the heating process. This means Loomino must cycle through temperatures of 95 degrees C and 37 degrees C with relative accuracy and speed. In order to accomplish the heating process, Loomino is equipped with a heating cartridge that quickly raises the temperature of the reaction chamber. A thermistor is attached to regulate the temperature and provide feedback for the system.</p>
 
<p>De Novo Synthesis requires the removal of a heat-labile group at 95 degrees C. The reaction chamber must then be cooled in preparation for the next reagent for the heating process. This means Loomino must cycle through temperatures of 95 degrees C and 37 degrees C with relative accuracy and speed. In order to accomplish the heating process, Loomino is equipped with a heating cartridge that quickly raises the temperature of the reaction chamber. A thermistor is attached to regulate the temperature and provide feedback for the system.</p>
  
 
<p>Once the reaction has completed in the heating step, the cooling process begins. A thermoelectric cooler is turned on, rapidly cooling the reaction chamber. A stock fan and heat sink are attached as well to assist in cooling and providing airflow through the machine. Once the machine drops to the proper temperature, the cooling stops. The thermistor continues to provide feedback and regulate the temperature.</p>
 
<p>Once the reaction has completed in the heating step, the cooling process begins. A thermoelectric cooler is turned on, rapidly cooling the reaction chamber. A stock fan and heat sink are attached as well to assist in cooling and providing airflow through the machine. Once the machine drops to the proper temperature, the cooling stops. The thermistor continues to provide feedback and regulate the temperature.</p>
  
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<h2>
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<p>Once De Novo Synthesis has completed, the machine is able to run the same thermal cycling process as a PCR machine. Loomino is able to cycle between 95 degrees C, 55 degrees C, and 72 degrees C for the standard PCR cycle. As it is electronically controlled, any number of cycles is possible. </p>
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<h2>Electronics</h2>
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<p>Loomino is controlled using an Arduino microcontroller. It is equipped with motorshields to send the proper signals to control the stepper motors. The system is preprogrammed to initiate the proper procedure to run de novo synthesis. The only constant variation control is in the thermal cycling. The thermistor uses variable resistance based on temperature to change the current running through the circuit. That current is then translated to a temperature. Using proportional-integral-derivative control, the Arduino is able to turn the heating cartridge on and off to remain at a stable temperature.</p>
  
 
<center><img src= "https://static.igem.org/mediawiki/2014/1/15/CooperUnionTdT1.jpg" width="500"/></center><br>
 
<center><img src= "https://static.igem.org/mediawiki/2014/1/15/CooperUnionTdT1.jpg" width="500"/></center><br>
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<h2> Project Description for Biological Components </h2>
 
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<p>The biology component of our project focused on two main concepts crucial to the functioning of the complete device.  The first of these was to test the idea of bonding DNA to a glass microscope slide as a means of preventing it from being lost in wash steps.  We also sought to characterize different variants of Terminal deoxynucleotidyltransferase (TdT) to find one best suited for the synthesizer.  A third but less pursued topic of study was the possibility of making a heat stable TdT variant using an intein system to circularize the enzyme.</p>
 
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We considered a number of chemical means by which one can bind DNA to glass [1].  The system we chose involved bonding DNA with a five prime thiol modified group to a silanized glass slide.  This method was chosen because the silanization process could be carried out in-house and adding a modified thiol group to the five prime end of DNA is a relatively inexpensive modification.  The methodology was based on a paper by Rogers et al. at Johns Hopkins [2].  After the reaction is complete, the DNA should be bonded to the glass by a disulfide bond.  To test that our glass slides had DNA on them, we would use ethidium bromide since it is known to attach to DNA strands.</p>
 
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In parallel with the glass slides, our team designed three mutated TdT sequences based on a paper by Repasky et al.[3]  In order to characterize these forms of TdT, we would have to ligate them into a high copy expression vector and then transform them into a cell line optimized for expression.  Protein purification of these cells would then follow.  Finally, we would have to test this protein by treating oligonucleotides with the TdT and running them on a polyacrylamide gel.  This would reveal whether or not the TdT can add base pairs to the free three prime end of a DNA molecule.</p>
 
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The intein based TdT system was the lowest on our priority list.  This is because it is only useful if the synthesizer utilizes heat-labile nucleotides.  Ideally, the final system will utilize U.V.-labile nucleotides which would make this system superfluous.  Despite this, we still worked on this angle of the project since U.V.-labile nucleotides are expensive.  In order to circularize TdT, we would have to design a linker sequence between the N and C termini of the protein.  This linker would be incorporated into the TdT sequence along with the intein system.  The remaining steps would be the same as those for our other TdT mutants the only difference being a heating step to insure that the enzyme is heat stable.  </p>
 
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<h3>References</h3>
 
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<p>1. Strategies for Attaching Oligonucleotides to Solid Supports. Integrated DNA Technologies (2014).</p>
 
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2. Rogers et al. Immobilization of Oligonucleotides onto a Glass Support via Disulfide Bonds: A Method for Preparation of DNA Microarrays. Analytical Biochemistry 266, 23–30 (1999).</p>
 
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3. Repasky et al. Mutational Analysis of Terminal Deoxynucleotidyltransferase Mediated N-Nucleotide Addition in V(D)J Recombination. Journal of Immunology 172(9):5478-88 (May 2004).</p>
 
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Revision as of 05:59, 18 September 2015

Cooper Union 2015 iGEM



What is Loomino?

Loomino is a simple DNA synthesizer that is built using open-source software and hardware. Its mission is to reduce the amount of time a lab would need to wait for synthesized DNA to arrive. Current DNA synthesizers use phosphoramidite-based procedures with hazardous chemicals and dangerous waste. It is highly uncommon that a lab outside of large corporations would be able to perform DNA synthesis, and therefore this process slows down bioresearch.

The machine itself has 3 subsystems: fluidics, thermal cycling, and electronics. The main criteria we were aiming for was ease of use, and feasibility. This criteria was used to judge the design, and optimize the machine.

Fluidics

In order to properly add reagents to the reaction chamber, Loomino uses a series of peristaltic pumps to apply pressure thus creating flow. These peristaltic pumps are constructed out of a NEMA17 stepper motor, 3D printed parts, and machined bearings. As peristaltic pumps are expensive, we decided that an open source design would be best suited for Loomino. The overall cost of such a pump is far cheaper than purchasing a prefabricated pump from a manufacturer.

Another advantage of using a peristaltic pump is that Loomino has the ability to draw air into the tubes. This allows for a gas buffer between the fluids which means we are able to move accurate volumes of fluid without excess waste. A mechanism is able to lower the reagents such that the tubes are not submerged in fluid, allowing for us to draw air. Without such a mechanism, large amounts of reagent would be wasted during the wash process which reduces the cost efficiency of the device.

There were many alternatives to create fluid flow such as using a system of syringes, or having a gas pressure microfluidic device. The advantage of having a syringe system is that the mechanism needed to pump is relatively simple. By rotating a threaded rod with a motor, we could create linear motion that would compress a syringe, thereby pumping fluids. The issue is that it lacks the ability to pump air, and thus we would encounter large amounts of waste. Also, a primary concern is accuracy. As the stepper motors are able to move within a 1.8 degree rotation excluding half-steps, we are able to more finely control flow rates. A microfluidic device requires more equipment either for lithography or molding PDMS. A gas pressure system would also need to be implemented, and therefore we determined these to be not feasible.

Thermal Cycling

De Novo Synthesis requires the removal of a heat-labile group at 95 degrees C. The reaction chamber must then be cooled in preparation for the next reagent for the heating process. This means Loomino must cycle through temperatures of 95 degrees C and 37 degrees C with relative accuracy and speed. In order to accomplish the heating process, Loomino is equipped with a heating cartridge that quickly raises the temperature of the reaction chamber. A thermistor is attached to regulate the temperature and provide feedback for the system.

Once the reaction has completed in the heating step, the cooling process begins. A thermoelectric cooler is turned on, rapidly cooling the reaction chamber. A stock fan and heat sink are attached as well to assist in cooling and providing airflow through the machine. Once the machine drops to the proper temperature, the cooling stops. The thermistor continues to provide feedback and regulate the temperature.

Once De Novo Synthesis has completed, the machine is able to run the same thermal cycling process as a PCR machine. Loomino is able to cycle between 95 degrees C, 55 degrees C, and 72 degrees C for the standard PCR cycle. As it is electronically controlled, any number of cycles is possible.

Electronics

Loomino is controlled using an Arduino microcontroller. It is equipped with motorshields to send the proper signals to control the stepper motors. The system is preprogrammed to initiate the proper procedure to run de novo synthesis. The only constant variation control is in the thermal cycling. The thermistor uses variable resistance based on temperature to change the current running through the circuit. That current is then translated to a temperature. Using proportional-integral-derivative control, the Arduino is able to turn the heating cartridge on and off to remain at a stable temperature.