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<p style="font-size:64px"><b>Project Design: Hok/Sok</b></style>
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<p style="font-size:64px"><u><b>CHIP: UMD's homemade PCR</b></u></style>
 
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<h1><b>Hok/Sok Construct</b></h1>
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<h1><b>What is PCR?</b></h1>
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<p>
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<li> 1. Full Hok-Sok sequence taken from Gerdes et al. Sequence is originally from the R1 plasmid of <i>E. coli K-12</i>.</li>
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Polymerase Chain Reaction or PCR is a common tool used in the field of biology to amplify DNA or RNA. Invented by Dr. Kary Mullis, PCR is conducted trough cycling DNA, primers and enzyme through various temperatures. Generally starting with a value near 95 degrees Celsius; used to break the Hydrogen bonds between double strands a process called denaturation. The machine then cools down to annealing temperature, with values near 50-60 degrees, at this point primers are able to attach to the template strand of DNA. This stage is then followed by extension temperature, around 72 degrees, at this point the polymerase is able to extend and add nucleotides to the primer.
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<li> 2. g-Block of Hok-Sok sequence ordered from Integrated DNA Technologies, then assembled into pSB1C3 using the Gibson method.</li>
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<a name="HS"><h1><b>Fluorescence Studies</b></h1></a>  
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<h1><b>Purpose</b></h1>
  
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<li>1. Hok-Sok was coupled to a constitutive generator of unstable RFP to form a larger composite part.</li>
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Although the process of amplifying genetic material is remarkable, the hardware needed to do it is relatively simple-- all that is required are three different temperatures which are maintained by the machine, enabling the enzymes and template to do the work of PCR. Current PCR machines cost thousands of dollars, and although there exists open source, DIY PCR machines, their costs still range in the hundreds of dollars. Here at the University of Maryland, we thought that that was an absurd notion. PCR, because of its simplicity and utility, is a robust tool for the diagnosis of many diseases both in the developed and developing world. Making the device cheaper would give more people accessibility to this platform. Accessibility enables further innovation and development of novel methods for disease detection and this in turn enables better and faster diagnosis and treatment both in the developed and developing world.  
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<li>2. Unstable LVA-tagged RFP has a half-life of 1 hour and allows for more current measurements of protein production.</li>
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<br> </br>
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<li>3. For our first testing method to see if Hok-Sok was capable of maintaining a plasmid, we wanted to measure how RFP fluorescence was retained over many generations in two <i>E. coli</i> strains: BL21 and DH5α.</li>
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Another major advantage of "cheap" is education. Here at the University of Maryland, we acknowledge that iGEM is a competition, however we also understand that this competition is also a collaboration. It is an opportunity for all of us to learn from one another and serves as the foundation for future discovery, innovation, and new projects. We hope that our work with the PCR machine will inspire many more teams to tackle designing hardware. We hope that our current collaborations with Duke University foster better and more innovative projects from both of our teams. And most important, we hope that our efforts will be able to inspire the future generation of iGEMer's and the newest members of the iGEM community; high school students.
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<li>4. In order to test, five sets of cultures were grown in LB media for fluorescence studies.</li>
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I remember, along with my fellow teammates, learning about PCR by cutting up little paper nucleotides and putting them into a brown bag and then having our hands act as the "polymerase" that would pluck the nucleotides out and match them with the template strand we were given. I remember taking away very little from this "lab" other than a few paper cuts. In subsequent years, I went through a few internship programs where I was able to learn in greater detail the steps of PCR, eventually learning how to design primers, program the machine, and setup my own reactions. However, I believe that if we truly want to bring synthetic biology to the public, we have to allow them the opportunity to actually do PCR, not through a paper bag which is conceptual understanding, but a real reaction where the end products are the real deal, actual amplified DNA. We still have a ways to go... the enzymes have to become cheaper pipettes need to become cheaper, but designing a below 50 dollar PCR machine is the first step in this endeavor.                  
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<li>A. Constitutive unstable RFP grown <b>with</b> chloramphenicol (33 µg/mL) in media <b>(+ Control)</b></li>
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<li>B. Constitutive unstable RFP grown <b>without</b> chloramphenicol</li>
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<li>C. Unstable RFP without promoter <b>with</b> chloramphenicol <b>(- Control)</b></li>
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<li>D. Hok-Sok + Constitutive unstable RFP grown <b>with</b> chloramphenicol</li>
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<li>E. Hok-Sok + Constitutive unstable RFP grown <b>without</b> chloramphenicol</li>
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<li> 5. Cultures were grown for 20 hours prior to fluorescence measurements.</li>
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<li> 6. Plate reader was used for fluorescence measurements.</li>
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<li>A. 200 µL of undiluted culture was pipetted into each well.</li>
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<li>B. Excitation wavelength was 555 nm. Emission wavelength was 584 nm.</li>
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<a name="PCR"><h1><b>Plating Studies</b></h1></a>  
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<h1><b>C.H.I.P: Cheap Homemade Innovative PCR</b></h1>
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<p>Along with measuring culture fluorescence, we also tested the ability of hok-sok to maintain a plasmid using daily chloramphenicol challenges. The protocol is as follows:</p>
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<p> Our first design for C.H.I.P. was modeled after a more conventional PCR machine design. This first prototype used two peltier units, stacked on top of each other, to heat a customized aluminum block that sat on top of the two units and held the PCR tubes. In order for our system to have feedback, we embedded a temperature sensor in the aluminum block to detect the temperature of the wells that held the PCR tubes. The sensor then reported back to an Arduino UNO, which then regulated the energy flow to the peltier units, thereby heating and cooling the block and tubes while closing the control loop. However, after much testing, this design proved to be unoriginal, expensive, and inefficient. While the conventionality of the design itself did not pose an issue, we realized that the parts used to assemble it were not as well-known or easily accessible to the general public, which we felt would take away from the possible applications of this machine. In addition, although the price of this first prototype was relatively inexpensive in contrast to laboratory grade PCR machines, the price still ranged in the hundreds of dollars. Lastly and most importantly, the greatest issue with our design was the inefficiency of the hardware; we found that the peltier units were not able to cycle through the desired temperatures fast enough, e.g., the unit would take 5 to 10 minutes just to rise up to 95℃. After considering all of these factors, we began a redesign of C.H.I.P. to better suit the needs of the “Do-It-Yourself” market. </p>
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<li>1. Dilute each culture (1 : 10<sup>6</sup>) with LB media</li>
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<p> The idea for our current thermocycler design first came into form when we found that our original prototype was not ramping up to the desired temperatures fast enough. Because of this problem, we looked into other options for heating the machine and, in the process, disassembled a hair dryer to find out how the heating mechanism worked. To our pleasant surprise, we found that the hair dryer was able to reach very high temperatures—much higher than the desired maximum of 95℃ for PCR—in a matter of seconds. We then made a decision to suspend construction on the peltier-centered thermocycler in order to see how successful we could be and how far we could go with making a rapid PCR machine out of a hair dryer. Before this decision, we took into consideration the danger of working with a hair dryer, failure due to uncertainty that the machine could be effectively controlled, and, on top of that, having less time to work on it. Nevertheless, we took the risk and are pleased to show you the results of our efforts—the creation of C.H.I.P. 
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<li>2. Plate 10 µL of each diluted culture on a LB-agar + chloramphenicol plate</li>
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<li>3. Incubate plates overnight</li>
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</p>The goal in doing this was to determine how many bacteria were surviving by retaining their plasmids. We took notice of the color of the colonies and the number of colonies on the plate.</p>
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<p>For continuing generations of BL21 strain E. coli, we observed that on the plates for groups A and B, there was growth but no redness. If the bacteria were retaining the plasmids with the chloramphenicol resistance, the RFP gene should have been expressed and the colonies should fluoresce. We hypothesized that the chloramphenicol resistance gene was being recombined into the bacterial genome so the bacteria could therefore freely eject our inserted plasmids. As BL21 carries the gene for recombinase, it is possible. However, DH5α, as a common cloning strain, does not have recombinase. We created a new generation with every group (A, B, C, D, and E) to test whether the same plate would have similar results or once the bacteria stopped fluorescing, there would be no growth on the plates.</p>
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<a name="PCR"><h1><b>Growth Curve</b></h1></a>  
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<h1><b>C.H.I.P.'s Design</b></h1>
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<p>We created a growth curve of Hok/Sok in comparison to controls to test the effectiveness of the Hok/Sok system in keeping the bacteria alive. We had four groups:
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<p>The design of CHIP started when we bought a hairdryer in the hopes of using the heating unit as part of our first PCR machine. However, as we were dismantling and testing the hairdryer, it became apparent to us that the heating system inside the hairdryer could reach the necessary temperatures independent of the peltier units already in use. With this in mind, we began working out how to wire the hairdryer so that we could regulate the heating unit and the fan separately.
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<li>• Hok/Sok without chloramphenicol</li>
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After a lot of soldering and reworking the internal safety measures inside the hairdryer, we were able to wire the system so that we could turn the heat on and off while running the fan continuously. Using autoclave tape, we secured a sheet of aluminium foil to the top of the heating unit of the hairdryer. The outer casing of the hairdryer had been removed. We placed a heat sensor inside the tin to measure the temperature of the air inside the machine.  By wiring the heat sensor to the arduino we were able to receive input/feedback from the sensor and adjust heating of the device to maintain our desired setpoints. We were able to regulate the heat of the machine and CHIP now thermocycled.
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<li>• Hok/Sok with chloramphenicol</li>
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<li>• RFP without chloramphenicol</li>
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At this point, we tried to perform our first PCR reaction, unfortunately we soon found that we had melted our tube. We learned that the machine had difficulty with evenly distributing the heat, since the tin foil was a rudimentary cover with holes punched into it without a proper understanding of what these holes would do to the heat distribution(see picture below). To better distribute the heat we removed our tinfoil lid and replaced with with a soda can. This can was designed with evenly spaced holes enabling for better heat distribution. Although we did not and still have not modeled the heat transfer of between the can's surface and the convection heating generated by the hair dryer, we were able to experimentally conclude that the heat distribution was more even across the can than the tin foil. For a better understanding we are currently in the process of modeling the heat transfer within the can to better design the apparatus.We are also in the process of milling aluminum with certain specifications in order to better regulate heat transfer. 
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<li>• RFP with chloramphenicol</li>
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<p>We started growing 250 mL cultures and monitored the OD at 600nm using a spectrophotometer over the span of 7.5 hours. </p>
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<p>After construction of the can based cover we tried PCR once more and still found that the reaction did not occur. We assumed that the heat sensor might have been an issue,; the sensor was exposed to the convected air and was relaying information about the air temperature instead of the temperature inside of the PCR tubes. This meant that our feedback system was not accurately responding and controlling the temperature inside of the PCR tubes. Assuming the temperatures inside the machine were not representative of the temperatures inside the PCR tubes, we put the heat sensor inside a PCR tube with mineral oil and placed this inside one of the holes. We ran another PCR reaction, ran the products on a gel and saw a large band of the correct size, indicating that CHIP worked.
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<img src="https://static.igem.org/mediawiki/2015/5/5a/IGEM_2015_PCR_wire_schematic.png" style="width:800px;height:588px;">
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<a name="PCR"><h1>Parts Referenced</h1></a>  
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<h1><b>Problems and Current issues </b></h1>
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<p> We have had one successful amplification with our machine however we understand that repeatability is a vital component of all lab work and currently we are attempting to make our device repeatable. From our early days of testing we found that peltier units were not powerful enough to enable PCR tube to reach 95 degrees. Although conventional PCR machines use these units frequently they are often specialized and tailored made to perform PCR. With this tailoring comes a high price tag that does not suit the DIY market, and so we found a solution in the form of a hairdryer. The fan and heating element of a hairdryer provide a control scheme that enables for cycling of temperature rapidly and accurately and they are relatively inexpensive. We have found that developing a housing for the PCR tubes and enabling even heat distribution is challenging. We often have found that our temperature sensor and the pcr reaction tube are not at the same temperature and degree of difference is a delta of over 10 degrees celsius. We are currently working of milling a block of aluminum with better and more consistent heat transfer properties, and modeling the heat transfer within the can. Our ambition is that this will enable better control of temperature within the device.        
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<li><a href = http://parts.igem.org/Part:BBa_K1783001>Hok-Sok Type I Toxin-Antitoxin Cassette</a></li>
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<p><b> References </b></p>
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<li>1. Gerdes, K., Thisted, T., & Martinussen, J. (1992). Mechanism of post-segregational killing by the hok/sok system of plasmid R1: Sok antisense RNA regulates formation of a hok mRNA species correlated with killing of plasmid-free cells. <i>Molecular Microbiology</i>, 223(1), 1807-1818. doi:10.1016/0022-2836(92)90714-U</li>
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<li>2. Mitsuoki Kawano (2012) Divergently overlapping cis-encoded antisense RNA regulating toxin-antitoxin systems from E. coli, <i>RNA Biology</i>, 9:12, 1520-1527, DOI: 10.4161/rna.22757</li>
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Revision as of 19:03, 17 September 2015