caption="Figure 3. A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes)"
caption="Figure 3. A: eRCA with 40.8 pM miR-1; B: negative control (no enzymes); 2% agarose gel ran for 1 hour at 48V"
/>
We then characterized and quantified the RCP from the eRCA reaction through the Lettuce reporting mechanism (see [Experiments: eRCA Readout](https://2023.igem.wiki/lambert-ga/experiments/)). Resultant fluoresence was quantified in a plate reader at exciation wavelength of 480 nm and emission intensity at 528 nm. The triplicate of eRCA with 40.8 pM of miR-1 exhibits significantly more fluorescence than that of the negative control (no enzyme), indicating that the eRCA reaction was successful (see Fig. 4).
@@ -18,35 +18,25 @@ difficulties of tedious pipetting and inaccurate results. Last year, Rolling Cir
Amplification (RCA) reactions we ran took the majority of our time. This took important
research time away, limiting us from our full potential. Issues like these inspired
us to develop LabPilot, a frugal automated liquid handler that allows researchers
to focus time elsewhere from running lengthy reactions like RCA. Pipetting is a fundamental
laboratory technique used to transfer precise volumes of liquid from one container
to another. Consistent pipetting is essential for achieving accurate results, maintaining
data integrity, and facilitating advancements in synthetic biology research. However,
manual pipetting, the primary method utilized in most labs, limits throughput (the
amount of liquid passing through a pipette) and is easily susceptible to human error.
To resolve these issues, automated liquid handlers are designed to automate and streamline
the process of pipetting liquids in laboratory settings. Liquid handlers can be programmed
to perform a wide range of pipetting tasks with high precision, accuracy, and efficiency,
enabling high throughput experiments (Liquid Handling Automation Benefits). However,
current automated liquid handlers costing between \$5000 and \$300,000 are difficult
to use (Retisoft, 2021). To provide underfunded labs access to liquid handlers, Lambert
iGEM developed LabPilot, a frugal liquid handler made with 3D printable and accessible
parts that can accurately pipette based on direct user input (Hentz & Knaide, 2014).
Therefore, it enables research to be conducted in an efficient manner, filling an
essential gap in lab settings.
to focus time elsewhere from running lengthy reactions like RCA. Pipetting is a fundamental laboratory technique used to transfer precise volumes of liquid from one container to another. Consistent pipetting is essential for achieving accurate results, maintaining data integrity, and facilitating advancements in synthetic biology research. However, manual pipetting, the primary method utilized in most labs, limits throughput (the amount of liquid passing through a pipette) and is easily susceptible to human error.
To resolve these issues, automated liquid handlers are designed to automate and streamline the process of pipetting liquids in laboratory settings. Liquid handlers can be programmed to perform a wide range of pipetting tasks with high precision, accuracy, and efficiency, enabling high throughput experiments (Liquid Handling Automation Benefits). However, current automated liquid handlers costing between \$5000 and \$300,000 are difficult to use (Retisoft, 2021). To provide underfunded labs access to liquid handlers, Lambert iGEM developed LabPilot, a frugal liquid handler made with 3D printable and accessible parts that can accurately pipette based on direct user input (Hentz & Knaide, 2014). Therefore, it enables research to be conducted in an efficient manner, filling an essential gap in lab settings.
caption="Figure 4. Indentations on the bed plate for the molds to rest on."
/>
### XYZ Motor Axis
LabPilot utilizes five Nema 17 motors (Nema 17 Stepper Motor) (see Table 1) to control the three axes of movement needed for the pipetting mechanism. Nema 17 motors have a micro-stepping feature, enabling small and precise movements. Each motor is controlled by an Arduino Mega 2560 with a Ramps 1.4 CNC shield containing five A4988 motor drivers (see Table 1). The CNC shield uses two motors to control the vertical movement of the bed plate, as it is the heaviest moving part. The left/right movement and the forward/backward movement of the micropipette are controlled by one motor each. The last motor controls the pipetting mechanism and is placed above the micropipette. Additionally, we placed limit switches for each axis to prevent over-rotation of the motors and calibration to home coordinates similar to 3D printers. Each axis has linear steel rods that act as supports, with bearings in place to ensure the pipette mechanism slides smoothly across the rods. We attached timing belts (thin rubber belts) with teeth (see Table 1) to the motor to carry the pipette left, right, forwards, and backwards across the X and Y axes.
LabPilot utilizes five Nema 17 motors (Nema 17 Stepper Motor) (see Table 1) to control the three axes of movement needed for the pipetting mechanism. Nema 17 motors have a micro-stepping feature, enabling small and precise movements. Each motor is controlled by an Arduino Mega 2560 with a Ramps 1.4 CNC shield containing five A4988 motor drivers (see Table 1). The CNC shield uses two motors to control the vertical movement of the bed plate, as it is the heaviest moving part. The left/right movement and the forward/backward movement of the micropipette are controlled by one motor each. The last motor controls the pipetting mechanism (see Fig. 5) and is placed above the micropipette. Additionally, we placed limit switches for each axis to prevent over-rotation of the motors and calibration to home coordinates similar to 3D printers. Each axis has linear steel rods that act as supports, with bearings in place to ensure the pipette mechanism slides smoothly across the rods. We attached timing belts (thin rubber belts) with teeth (see Table 1) to the motor to carry the pipette left, right, forwards, and backwards across the X and Y axes.
In addition to the linearly moving axes, LabPilot requires a motor to press the micropipette to the first and second stops. The pipetting mechanism motor converts the rotational motion of the motor into a linear force that can push down on the micropipette button. The motor applies a fine-tuned force to achieve first and second stops. Currently, LabPilot is designed to be used with Eppendorf micropipettes to ensure everything fits correctly. We attached a stationary arm to the bed plate to eject the pipette tips. The micropipette moves into the arm while sequentially traveling upwards. This way, the tip is pulled off the micropipette and discarded underneath.
caption="Figure 6. shows how the user input on the app controls the microcontroller."
/>
The app provides instructions to the microcontroller via the Arduino serial USB connection (see Fig. 7). By utilizing a system where the app executes high-level operations by compiling pipette actions into low-level instructions for the microcontroller, users have complete control over their LabPilot device. They can stop or pause it at any time.
The app provides instructions to the microcontroller via the Arduino serial USB connection (see Fig. 6). By utilizing a system where the app executes high-level operations by compiling pipette actions into low-level instructions for the microcontroller, users have complete control over their LabPilot device. They can stop or pause it at any time.
All pipetting actions consist of a reagent source and a dispensing source. Users can click on any well or beaker to select a reagent source. After choosing a reagent source, users can select a dispensing source by clicking on any well/beaker or multiple dispensing sources (by clicking and dragging or holding “shift” while they select multiple sources). Afterward, users will select the amount they want to dispense to each dispensing source and can add it to the queue (see Fig. 7). All pipetting actions in the queue are performed in the order they were assigned but can be dragged around to reorder in the queue.
@@ -62,7 +62,7 @@ In addition to frugal technologies, Lambert iGEM aimed to decrease the cost of R
## Estrogen
While researching CAD risk factors and statistics, we came across literature that informed us about disparities among the sexes concerning early CAD diagnosis. We first conferred with Dr. Mindy Gentry, a women’s cardiologist, to get her opinion on the sex differences in cardiovascular health. She confirmed our insights about estrogen being a factor in increased susceptibility to CAD in women. To further our scientific research, we spoke to Dr. DeLisa Fairweather, a director of translational research in the Department of Cardiovascular Medicine at Mayo Clinic's Florida campus. She educated us on estrogen and its role as a CAD suppressor, and explained how women after menopause are more likely to develop heart disease because estrogen levels drastically decrease after menopause, causing women to be more likely to develop inflammation. Dr. Fairweather detailed that women who have inflammation in the heart are more likely to develop autoimmune disease and CAD. Dr. Fairweather not only gave us insight into estrogen and its correlation to women's risk for CAD. Additionally, she also guided us in the further development of our inclusivity by discussing how the hormone affects men as well. She told us that since men have a low estrogen level that also remains stable, they would be an ideal population to test for consistency in results, unlike women who have fluctuating levels of the hormone. Under the guidance of Dr. Fairweather, we tested miRNA-20b using Rolling Circle Amplification. In the presence of estrogen, CAD suppressors are activated and miRNA-20b is produced. miR-20b causes a negative feedback loop and suppresses estrogen, which will in turn inhibit the CAD suppressors. Dr. Fairweather explained how in men, a high level of miR-20b means that they are likely to develop CAD, however, in premenopausal women with inflammation symptoms, miR-20b protects them from developing CAD. She explained how if there is a high level of 20b, it will inhibit the estrogen from causing a catalyst between the developing autoimmune disease and CAD. We tested miR-20b with the RCA mechanism and ran a 1% gel to validate the presence of our product (see Fig. 5). Ultimately, increasing the factors that can be tested to signify CAD correlation that also encompasses a wider yet specified group allows for quality proactive care to be implemented. (See Wetlab: Inclusivity)
While researching CAD risk factors and statistics, we came across literature that informed us about disparities among the sexes concerning early CAD diagnosis. We first conferred with Dr. Mindy Gentry, a women’s cardiologist, to get her opinion on the sex differences in cardiovascular health. She confirmed our insights about estrogen being a factor in increased susceptibility to CAD in women. To further our scientific research, we spoke to Dr. DeLisa Fairweather, a director of translational research in the Department of Cardiovascular Medicine at Mayo Clinic's Florida campus. She educated us on estrogen and its role as a CAD suppressor, and explained how women after menopause are more likely to develop heart disease because estrogen levels drastically decrease after menopause, causing women to be more likely to develop inflammation. Dr. Fairweather detailed that women who have inflammation in the heart are more likely to develop autoimmune disease and CAD. Additionally, she also guided us in the further development of our inclusivity by discussing how the hormone affects men as well. She told us that since men have a low estrogen level that also remains stable, they would be an ideal demographi to test for consistency in results, unlike women who have fluctuating levels of the hormone. Under the guidance of Dr. Fairweather, we tested miRNA-20b using Rolling Circle Amplification. In the presence of estrogen, CAD suppressors are activated and miRNA-20b is produced. miR-20b causes a negative feedback loop and suppresses estrogen, which will in turn inhibit the CAD suppressors. Dr. Fairweather explained how in men, a high level of miR-20b means that they are likely to develop CAD, however, in premenopausal women with inflammation symptoms, miR-20b protects them from developing CAD. She explained how if there is a high level of 20b, it will inhibit the estrogen from causing a catalyst between the developing autoimmune disease and CAD. We tested miR-20b with the RCA mechanism and ran a 1% gel to validate the presence of our product (see Fig. 5). Ultimately, increasing the factors that can be tested to signify CAD correlation encompasses a wider yet specified group allows for quality proactive care to be implemented. (See Wetlab: Inclusivity)
