Since Theodor Escherich’s discovery of them in 1886, Escherichia coli has become a staple model species for study in bioscience laboratories. The species’ natural resistances, resiliencies, and attributes made them ideal for growing in lab conditions. With the sequencing of the E. coli genome in 1997, scientists gained the ability to manipulate the species at will. Although this focus on E. coli has aided in the rapid advancement in microbiology and synthetic biology, there remains a large gap in research regarding other non-model prokaryotes. Such organisms often exhibit unusual properties, but due to the technical barriers prohibiting their study, little research can be conducted. BHR Plasmid Kit seeks to destroy these barriers.
One of the central problems facing researchers is that plasmids, short circular pieces of DNA, are often nonfunctional in multiple different species of bacteria. Although metabolic load affects the lifespan of plasmids accurately in E. coli, additional variables must be accounted for in non-model species. This due to the fact that origins of replication, the segment of a plasmid or chromosome at which DNA replication begins, are often limited to a small clade of bacteria.
BHR Plasmid Kit is making an extensive catalog as to which genetic parts work in which species. Using these data, we are developing a standardized “kit” to help other labs work with non-model species. Such a kit would contain varieties of pre-assembled plasmids as well as their constituent parts. Through a series of design techniques and specialized parts, we can maximize the efficiency and ease-of-use of the kit.
To maximize ease of use, we insert multiple assembly plasmids into single eppendorfs. These may be used to transform into bacterial cells of the client’s choosing. A commonly asked question is that if multiple plasmids are used in the same reaction, how could a researcher decipher what plasmid was successfully transformed? The answer is that we equip our assembly plasmids with several selectable markers. These specialized parts allow a researcher to visualize, at a macroscopic level, the microbiological processes of his/her reaction. Antibiotic resistance genes will allow successfully transformed bacteria to grow on certain antibiotics such as chloramphenicol or kanamycin. Green fluorescent proteins (GFPs) or chromoproteins change the color of the transformed colonies — this can be viewed either by the unaided eye or with the help of a black/blue light. Finally, “barcodes” allow reliable and definitive identification through DNA sequencing. We include primers in the kit to sequence barcodes.
Our lab uses techniques that might seem foreign to other labs. Among the most important of said techniques is Golden Gate Assembly (GGA). GGA is a powerful cloning technique using Type IIs restriction enzymes to ligate multiple parts together in a single reaction. This can occur because Type IIs enzymes such as BsaI and BsmbI cut outside of their restriction sites — this allows us to “program” our parts with very particular overhangs. As many as ten parts can be assembled at once.
The BHR Plasmid Kit allows microbiologists and synthetic biologists to work with species previously thought to be out-of-reach. The applications of the kit are endless: modifying cyanobacteria to break down BPA and even programming symbiotic bacteria to add nutritional value to kombucha. The BHR Kit will help us assemble a better future.
Part types 1, 2, 4, 5, 6, 7 and 8 are important for plasmid replication. Part type 3 is the plasmid part/gene to be incorporated in the non-model organism. Part type 8b helps maintain a plasmid. When a non-host organism needs to be transformed, several assembly plasmids of different genetic parts can be transformed into the microorganism. The plated surviving organisms’ DNA can be sequenced to reveal which assembly plasmids can be maintained and replicated.