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Project Description

When a cell makes protein, it is easy enough to leverage the protein itself to see if the gene to make it is present. However, in all genes, the common component in the genetic code that makes it up. Is it possible to leverage the ease and predictability of nucleic acid interactions to better screen for proteins? Is it possible to signal for non-coding and knocked-out DNA sequences. In dCas9, we saw a system.

With dCas9, Duke iGEM 2014 saw an effect we called decoy binding where a fixed concentration of dCas9 will be lured away from a reporter gene by the presence of the same sequence elsewhere in the cell. As the number of decoys increases, the repressive force of dCas9 weakened on the reporter gene. In this phenomenon, we saw rudimentary signally based not on protein concentration but on DNA concentration. We saw that this behavior was worth investigating both at a basic level and then for use in applications.

We at Duke iGEM saw an opportunity of leverage the phenomenon of dCas9 decoy binding, studied as last year’s project, to directly signal the presence or absence of a set genetic sequence into the induction of a gene on a different operon. We hope that this construct allows for more versatility in recognizing a gene or non-coding region in the creation of more complex gene circuits. In order to highlight the capabilities of the circuit, we wanted to use it in the context of one of the most dangerous gene developments in modern history: antibiotic resistance.

One of the greatest discoveries since the germ theory of disease was the discovery of chemicals that could counter them. Penicillin and other drugs helped save countless lives and revolutionized the way we saw sickness. But in 1947, four years after the initial mass-production of penicillin, the first resistances began to appear. Already, certain drugs like penicillin have fallen out of medical utility. The problem is only expected to become worse with fewer antibiotics being developed and resistance occurring for these drugs faster and faster. The CDC reports MRSA, multidrug-resistant tuberculosis and other antibiotic resistant bacteria kill 23,000 a year[1] in the US alone.

The reason for this spread is obvious, with each dose of antibiotic putting massive selective pressure on the antibiotic gene. Especially when an antibiotic regiment is left uncompleted, those bacteria resistant to the antibiotic will survive to repopulate. With each prescription of antibiotics, this effect is repeated until full populations are filled with the resistance. Non-medical antibiotic usages within agricultural waste and cleaning products have only exacerbated the problem.

But what if the game of growth rates was shifted to favor the non-resistant bacteria? Naturally, the favored solution would be to also kill antibiotic resistant during treatment, but until then, the next best solution may be a weaker and continual pressure targeting these genes themselves. This pressure will give the antibiotic susceptible bacteria an opportunity to regain a majority of the population between antibiotic treatments. These slow “resets” could buy time for the development of new drugs or allow for the recycling of old drugs previously left ineffective.

We attempt to provide a negative selective pressure through the induction of programmable cell death genes. We hope to produce a plasmid to transform, conjugate, and in the future transfect into bacteria that provides the necessary machinery for limiting the populations of only antibiotic-resistant bacteria. In this first stage of our project, we find a suite of programmable cell deaths in order to find a gene both effective on bacterial populations while maintaining complete safety for eukaryotic and especially human cells.

We created adorable CELL DEATH GENES to terrify our ANTIBIOTIC RESISTANT BACTERIA.

Overview

The idea to design and build a thermocycler that could be built with parts found in and around the house was planted in our heads by our advisors. When an old PCR machine was salvaged from Duke Surplus, that idea became ambition. Was it even feasible for a team of biologists to create a machine that sold for upwards of $2000 for less than $200 and with parts that could be found for free or on Amazon? A minimal amount of parts are required to make a PCR machine functional. Behind the heating and cooling of the PCR tube well (called “heating block”) is a Peltier chip. A Peltier chip’s function is quite simple – it heats up rapidly by stealing heat from the other side of the chip when current is sent through one way, and cools down rapidly when that current direction is reversed. The component responsible for controlling the direction of the current is an H-Bridge, which is very simply a parallel arrangement of four switches. Closing two switches causes the voltage from the DC power source to be defined one way while closing the other two switches causes the voltage to be switched. A thermistor or thermocouple is required to monitor the temperature of the heating block. Both allow you to convert a voltage into a temperature reading. The brains behind the machine is the Arduino board, which is responsible for processing the current temperature of the heating block and telling the H-Bridge which direction to send the current to get the heating block to the desired temperature.

Acknowledgements

• Thank you to Ben’s dad, Patrick Hoover, for being able to find miscellaneous screws and capacitors and tools and the idea behind Prototype V2.
• Thank you to the University of Maryland iGEM team, with whom this project is closely tied. For further information on the extent of collaboration, read below.

Conceptualization

The original objective in designing a PCR machine was to design the kind of machine that anyone could look at the parts list for, buy whatever was needed online, and assemble it themselves according to a publicly designed blueprint. The goal, then, was akin to creating a DIY, college student rendition of OpenPCR’s open-sourced thermocycler design, which sells a 16 well thermal cycler for $650.
image here
This task proved incredibly daunting, and the mission statement soon became proving that a functional thermal cycler could be built from parts found around the house.

Part List

Prototyping began by disassembling an old desktop computer for parts. Parts salvaged from this are as follows:

• Power Supply – Capable of running 12A at 12V. Printed details can be crudely seen below. image here
• Heat Sink – For preventing the Peltier Chip from overheating on one side image here
• Two Fans – to keep heat sinks cool (visible partly in above picture) image here

The only part salvaged from the old 1994 thermal cycler found in surplus was the heat block, whose design has not changed.
image here
The rest of the parts were ordered from Amazon, and they include:

• Arduino mega2560
• Thermal compound (attaching Peltier to heat sink)
• VNH2SP30 Monster Motor Shield (H-Bridge)
• Aluminum Heatsink
• Peltier TEC1-12709

Prototype V1 – Shoebox Design (“The Testing Phase”)

image here
Thermistor Testing

The first design was a completely disorganized mess of temporarily attached cables and individual components that all needed to be tested. The first component that needed to be calibrated was the thermistor. A pack of 100 NTC thermistors were bought from Amazon for less than $7. They were calibrated by the Steinhart-Hart equation, which goes as follows:

Where A, B, and C are constants unique to each thermistor that can be calculated as a system of equations by taking the resistance of the thermistor at three different and known temperatures.

The thermistor used in the PCR machine was calibrated on a kitchen stove with multimeter capable of reading temperature through its own thermistor attachment.

Peltier Chip Testing

Before further prototyping, it became necessary to test the Peltier chip to see if it could reach temperatures of 95°C. The first TEC1-12706 (which can handle a maximum of 6A) could not reach the temperatures desired unless stacked, and when they were stacked were at high risk of shattering (cite: UMD iGEM team). With this foreknowledge, we went ahead and bought the TEC1-12709 model which is rated at 90W and can handle a maximum current of 9A (note: the openPCR project used a custom built Peltier chip that could handle a maximum of 86W and 7.8A. See openPCR.org BOM under Open Source Design). When plugged in to just the computer power supply, this new chip was drawing currents around 5A and heating a heating block twice as large as itself to 95°C in less than 1 min. Therefore, this chip has the power needed to conduct PCRs.

Motor Shield (H-Bridge) Testing

The first H-Bridge bought was an old L293D Arduino motor shield, capable of running the native voltage of the board (5V) and outputting 600mA per motor (max of 4 motors). We quickly realized this was an inadequate H-Bridge as we needed at least 5A for acceptable ramp up speeds. This problem was remedied when we came across the VNH2SP30 Monster Motor Driver on Amazon (Linked Here) which was rated for 14A of continuous current and up to 16V! The item was soon shipped and basic code implemented allowing the Arduino to control the voltage and direction of current through the PCR.

This Monster Motor Shield appeared at first to work great, as code was being tested to cycle through lower simulation temperatures continuously. It was able to make the Peltier reach 95°C many times, but it felt much slower than before. In the last week, we discovered that the website lied about the practical current use. The board shipped with a built in 4.12A current limiter. The unit also shipped with a defective VNH2SP30 H-Bridge. It failed to output a full 12V when the current was desired to flow in a counter-clockwise direction. Also, the shield has a safety to shut down all current when it senses potentially overheating. It has shut off countless times when it is just outputting 3A. For a unit rated at 14A, this is incredibly frustrating.

Brainstorming with UMD

Both of our teams decided separately that it would be cool to undertake the project of building a PCR machine for our team this summer. At the iGEM gathering hosted by the Maryland team at their university, we discovered the mutual progress. This started a series of scattered emails about updates and advice over the summer. While UMD sought novel and unique ways of solving the problems they ran into with weak Peltier chips, we stuck by our traditional H-Bridge and Peltier chip design as initial tests seemed very promising.

Summary

The Shoebox Design was a long-lived, frustrating phase of the prototyping process. Once each part of the machine was deemed suitable for incorporation into a more portable, permanent housing, the next phase of prototyping began.

Prototype V2 – Toolbox Design (“Functionality Phase”)

The goal of this design was to incorporate all the above components into a single compact and portable case. The case used was an old aluminum Remline toolbox my dad found sitting in the garage. After mounting the power supply to one side of the toolbox to maximize airflow in the case of a closed lid, the rest of the components were placed in a manner to balance the weight of the power supply without losing too much function. Unnecessary cables were tucked away. The rest of the cables were neatly organized and unnecessary lengths trimmed down. An aluminum heat sink was attached to the VNH2SP30 chips and another fan incorporated to try and keep the motor shield cool, but even this is insufficient for how hot those chips get.

images here


Current Status

We will be the first to admit that this design is still incredibly much a prototype. Right after assembling all the components into the toolbox, the heating block heated up to 95°C slowly but surely. After bringing the machine to college, where tools such as multimeters and oscilloscopes and soldering irons are reserved for classroom labs, the heating block refuses to cycle to 95°C. Sadly, we were not able to collect temperature over time data during the phase it was working. It can currently reach 78°C in just over a minute. We fear that assembling the H-Bridge into the toolbox has damaged it in some way, but have no way to test the voltage or current measurement.

Improvements

The one limiting factor in the success of the PCR machine is the Motor Shield. Replace this, and we have a truly functional thermal cycler in a toolbox. Therefore, the first thing to fix is to try and bypass the faulty and frustratingly fickle Motor Shield with a self-built H-Bridge incorporating real high current relays. These could be attached to a breadboard or soldered together on top of a prototyping PCB (Printed Circuit Board). This should solve both the current limiting problem and overheating safety shut off problem of the Motor Shield.

The thermistor really ought to be calibrated with a temperature sensor more accurate than a food thermometer. Smaller thermistors should be bought and multiple should be embedded into the heating block and the temperatures of each averaged to receive a more accurate temperature of the whole block.

We purchased the Arduino Mega2560 for this project. However, so many digital I/O pins are entirely unnecessary for this machine. The cheaper Arduino Uno R3 would be more than sufficient for the machine.

Once faster and more reliable heating is achieved, the code should be modified to enable fine controls such as controlling the ramp up rate and preventing excess overshoot of the desired temperature.

A heating lid should eventually be added so that condensation does not appear on the PCR tubes. This can be accomplished with a couple of 10W 10Ω power resistors in series, or even a couple 100W 1Ω power resistors in parallel (<$6 on amazon). The circuit from the heating lid could be wired in series with the Peltiers to prevent overloading the power supply.

The MRSA were getting too big for their britches.