Biochemical Pathways Download

Balanced expression of multiple genes is central for establishing new biosynthetic pathways or multiprotein cellular complexes. Methods for efficient combinatorial assembly of regulatory sequences (promoters) and protein coding sequences are therefore highly wanted. Here, we report a high-throughput cloning method, called COMPASS for COMbinatorial Pathway ASSembly, for the balanced expression of multiple genes in Saccharomyces cerevisiae. COMPASS employs orthogonal, plant-derived artificial transcription factors (ATFs) and homologous recombination-based cloning for the generation of thousands of individual DNA constructs in parallel. The method relies on a positive selection of correctly assembled pathway variants from both, in vivo and in vitro cloning procedures. To decrease the turnaround time in genomic engineering, COMPASS is equipped with multi-locus CRISPR/Cas9-mediated modification capacity. We demonstrate the application of COMPASS by generating cell libraries producing -carotene and co-producing -ionone and biosensor-responsive naringenin. COMPASS will have many applications in synthetic biology projects that require gene expression balancing.

In addition to multi-locus integration of Level 1 modules, COMPASS allows integrating completely assembled pathways (from a Level 2 Destination vector) into a single, defined locus of the yeast genome. Thereby, the position effect observed for transcriptional outputs of ATF/BS regulators positioned at multiple loci (Supplementary Note 1) is eliminated. This is important, when regulated expression of metabolic pathway genes is required.

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Further improvements may include the utilization of other inducers (e.g., light42), adding dynamic regulation to the system39, and adopting COMPASS to a wider range of hosts. In the present study, we used the COMPASS toolkit to optimize the production of biochemical compounds. However, COMPASS can facilitate many other projects in synthetic biology, including, e.g., the building of multisubunit protein complexes, the engineering of sophisticated gene-regulatory networks, or the construction of entire synthetic organelles. COMPASS thus has a great potential in many areas of synthetic biology.

From both inside and outside the body, cells are constantly receiving chemical cues prompted by such things as injury, infection, stress or even the presence or lack of food. To react and adjust to these cues, cells send and receive signals through biological pathways. The molecules that make up biological pathways interact with signals, as well as with each other, to carry out their designated tasks.

Biological pathways can act over short or long distances. For example, some cells send signals to nearby cells to repair localized damage, such as a scratch on a knee. Other cells produce substances, such as hormones, that travel through the blood to distant target cells.

These biological pathways control a person's response to the world. For example, some pathways subtly affect how the body processes drugs, while others play a major role in how a fertilized egg develops into a baby. Other pathways maintain balance while a person is walking, control how and when the pupil in the eye opens or closes in response to light, and affect the skin's reaction to changing temperature.

Metabolic pathways make possible the chemical reactions that occur in our bodies. An example of a metabolic pathway is the process by which cells break down food into energy molecules that can be stored for later use. Other metabolic pathways actually help to build molecules.

Gene-regulation pathways turn genes on and off. Such action is vital because genes provide the recipe by which cells produce proteins, which are the key components needed to carry out nearly every task in our bodies. Proteins make up our muscles and organs, help our bodies move and defend us against germs.

Signal transduction pathways move a signal from a cell's exterior to its interior. Different cells are able to receive specific signals through structures on their surface called receptors. After interacting with these receptors, the signal travels into the cell, where its message is transmitted by specialized proteins that trigger a specific reaction in the cell. For example, a chemical signal from outside the cell might direct the cell to produce a particular protein inside the cell. In turn, that protein may be a signal that prompts the cell to move.

Researchers are learning that biological pathways are far more complicated than once thought. Most pathways do not start at point A and end at point B. In fact, many pathways have no real boundaries, and pathways often work together to accomplish tasks. When multiple biological pathways interact with each other, they form a biological network.

Researchers have discovered many important biological pathways through laboratory studies of cultured cells, bacteria, fruit flies, mice and other organisms. Many of the pathways identified in these model systems are the same as, or are similar to, counterparts in humans.

Still, many biological pathways remain to be discovered. It will take years of research to identify and understand the complex connections among all the molecules in all biological pathways, as well as to understand how these pathways work together.

Researchers are able to learn a lot about human disease from studying biological pathways. Identifying what genes, proteins and other molecules are involved in a biological pathway can provide clues about what goes wrong when a disease strikes.

For example, researchers may compare certain biological pathways in a healthy person to the same pathways in a person with a disease to discover the roots of the disorder. Keep in mind that problems in any number of steps along a biological pathway can often lead to the same disease.

Researchers currently are using information about biological pathways to develop new and more effective drugs. It likely will take some time before we routinely see specifically designed drugs that are based on information about biological pathways. However, doctors are already beginning to use pathway information to choose and combine existing drugs more effectively. 006ab0faaa