The Motif Discovery function of MEME Suite recognizes recurring and unbroken patterns in RNA or DNA sequences, referred to as “motifs”. Using this technology, we were able to identify conserved regions of bacterial 16s sRNA sequences for our target pathogens. This information helped us design primers that target the conserved motifs, thus allowing for the detection of that pathogen in water samples.

New England Biolabs (NEB) provides multiple cloud-based primer designing tools, each designed for different reactions. We used the NEB LAMP Primer Design Tool to obtain DNA sequences for the LAMP primers. The primers can easily be generated by inputting the sequence to be amplified and adjusting parameters such as ion concentrations, primer lengths, and melting temperature to fit the LAMP reaction.

Like NEB LAMP Primer Design Tool, OptiGene LAMP Designer is also capable of producing LAMP primer sequences using a downloadable software. OptiGene has additional abilities such as performing a BLAST search to avoid homologous sequences that could potentially result in incorrect sequence amplification. By using a second program with more complex capabilities to generate primers, more options were available to us to optimize primers requires for all LAMP reactions.

New England Biolabs (NEB) provides multiple cloud-based primer designing tools, each designed for different reactions. We used the NEB LAMP Primer Design Tool to obtain DNA sequences for the LAMP primers. The primers can easily be generated by inputting the sequence to be amplified and adjusting parameters such as ion concentrations, primer lengths, and melting temperature to fit the LAMP reaction.

PyMOL, maintained and distributed by Schrodinger, is a popular open source, 3D molecule modelling program among the scientific community. It allowed for the production of highly detailed 3D images of both small molecules and larger biological macromolecules, such as the proteins that played a key role in our project. Our team primarily used PyMOL to model Bst DNA polymerase and similar proteins, Sac7e and other DNA-binding proteins, and protein linkers such as the (GGGGS)4 linker we used to fuse the proteins together. All models of our full modified Bst polymerase, including point mutations and protein fusion using a linker were designed on PyMOL as well. These models allowed us to visualize the similarity between different proteins, thus allowing us to infer what challenges would need to be overcome when designing a modified polymerase. PyMOL was used extensively throughout the course of our project, beginning from developing a strong understanding of the proteins we are working with, to visualizing our final modified polymerase.

YASARA is another molecular modelling program, with high simulation capabilities. It has a user-friendly interface and allows a strong level of interaction with the protein. Its uses are similar to PyMOL, as both are molecular modelling programs, but we used YASARA with a focus on determining specific protein properties. The FoldX plugin was added to YASARA to gain access to tools that improve prediction of protein structure, functional domains, and catalytic sites. With YASARA, we analyzed the thermostability of Bst DNA polymerase and were able to choose three residues in the thumb of our polymerase to mutate in order to improve thermostability. We continued to analyze the modified protein to ensure that a significant improvement was made with these mutations.

As an AI system, AlphaFold is a program especially designed to predict the 3D model of protein structures with high accuracy. This program allows for the prediction of protein folding, shape, and secondary structure. With AlphaFold, we visualized how our fully modified Bst polymerase folds in real space, and how the addition of a DNA-binding protein would affect its binding affinity for a DNA template. In this way, were ensured our modified protein did not lose any of its functionality with the alterations we introduced.

GROMACS is an open source molecular dynamics software used for simulations of protiens, lipids and nucleic acids. We used GROMACS to perform molecular dynamic simulations on our fusion protien and its constructs to ensure each parts would be stable under aqueous conditions.

Benchling is a cloud-based platform that has a diversity of software to assist in biotech research and development, ranging from creating labels and barcodes to CRISPR design. While Benchling is commonly used for data management and collaboration, we primarily used Benchling to simulate restriction enzyme digestion of our DNA plasmid to ensure that our DNA sequence didn’t contain any accidental restriction enzyme cut sites. We were able to detect two cut sites that if left unchecked, would have resulted in undesired cuts in our Bst gene sequence.

Seaview is a software that can be used to graphically edit multiple sequence alignments. We used Seaview to align amino acid sequences of related proteins to assess their similarity. This was critical for determining conserved sequences are between different proteins and organisms. In addition, we aligned the variations of gene sequences of our modified Bst generated from different codon optimizing programs. This allowed us to identify the most reliable codon optimizer program and visualize the discrepancy codons of an optimized gene sequence.

The Codon Optimization Tool from Integrated DNA Technologies (IDT) adjusts the DNA sequence of one organism into a sequence that can be best expressed in a different organism. Initially, when submitting our modified Bst gene sequence, it was rejected to its high complexity. Thus, we used IDT’s codon optimizer to generate a new gene sequence that would still code for the same amino acid sequence. We found IDT to provide the most consistent sequence with the lowest complexity rating, so we used this program to determine Bst polymerase’s gene sequence (naturally found in the bacteria G. stearothermophilus) that is best expressed in E. coli K-12.

Solidworks is a 3D modelling software that allows for users to design and test 3D models and structures that can then be carried out and physically designed and used. The software helped us test fit parts together to make sure that they fit, as well as to help tolerance parts for manufacturing. It also helped us visualize what our kettle device was going to look like and let us decide whether certain models would be the best fit for the end product.

Arduino is a software and hardware project that lets users design circuits and control them using microcontrollers. It allows for hardware such as LED’s, temperature sensors, pressure sensors, buzzers, motors and more to be controlled and manipulated though the Arduino IDE on any computer. For our kettle device, we used a circuit comprised of LED’s, a temperature sensor, resistors and a microcontroller to monitor the temperature of the Pcr test so that it can remain at a constant 65 degrees Celsius.

The atom.io IDE is an open-source code editor that allows for multiple coding languages such as HTML, CSS and JavaScript to be written and edited with ease. For the construction of the team wiki this year, we used atom to code every single HTML, CSS, and JavaScript file needed to complete the team website successfully.

Prusa slicer is an application which allows for 3D models to be converted to Gcode and then exported to a Prusa Slicer 3D printer for printing. The program served as the way to export all of our 3D models for 3D printing and allowed us to adjust build settings such as infill percentage, orientation, and more.

Tinkercad is a program mainly used for 3D modelling. For our project, we used Tinkercad to test and analyze our circuits before building them to make sure that they were safe and working properly.