- What’s on Nucleus?
- Synthetic Cell
- “Hello, world!”
- Developing with synthetic cells 🛠️
Synthetic cells are a powerful tool for defined, scaleable synthetic biology. Building a synthetic cell from scratch includes three main processes:
- Assembling a cytosol
- Adding modules (encoded in DNA) to the cytosol
- Encapsulating the cytosol in a lipid membrane
Nucleus exists to provide a single, integrated source for open protocols, materials, and tools supporting the execution of these processes. Nucleus v0.1 supports construction, measurement, and testing of the PURE system; expression of several reporter modules; and liposome encapsulation, measurement, and analysis.
What’s on Nucleus?
We divide each Nucleus release into four broad categories:
Container: the means to separate the inside of a synthetic cell from its environment.
Cytosol: the mixture within a synthetic cell.
Content: biological programs which operate in the synthetic cell.
Synthetic Cell: containers, cytosols, and content combined together (integrated) into a particular working cell.
You can read more about each category below. For a more detailed discussion of the current status of Nucleus and its contents, please see
Containers hold the interior of synthetic cells (the cytosol, DNA content, and anything else), separating them from the outside environment. The act of putting these interior contents inside a container is called encapsulation. There are many types of containers and multiple ways to encapsulate things for any given type of container.
Nucleus currently includes one type of container: lipid vesicles (or liposomes).
Cytosols are aqueous mixtures of all the molecules that you want to be inside your cell. Cytosols can be very simple (e.g., water plus dye) or extraordinarily complex (e.g., cell lysate). Cytosols provide the molecular machinery and resources for a synthetic cell to work. In order for your cell to do what you want, you have to pick the right cytosol.
Nucleus includes one biologically active type of cytosol: PURE, a cell free expression system built from individually purified components. Use this cytosol if you need DNA translated to proteins or transcribed to RNA. DNA plasmids encoding the thirty-six proteins composing the PURE system, as well as plasmids which operate in PURE, are included in the Nucleus v0.1.0 release.
Additionally, Nucleus supports one inert prototyping cytosol, useful for practicing liposome encapsulation: water + HPTS, a fluorescent dye (see
Content here means two things. In the abstract design sense, content includes all the functions you can use to augment your synthetic cells. Nucleus modules are specific implementations of those functions that you can add to your cells. Concretely, content also means all the DNA that explicitly implements those modules. All DNA content on Nucleus is open source and released under the OpenMTA, meaning you can share and remix these sequences as you see fit, for personal, academic, or commercial use.
Nucleus v0.1.0 includes two detector modules: lac, a lactose responsive expression system; and tet, a tetracycline responsive system.
Additionally, Nucleus includes a collection of generally useful open source DNA parts, including protein reporters (fluorescent and colorimetric), bacterial promoters and translation initiation sites for building new modules, and an open source plasmid backbone called pOpen. All these parts are designed to be composable with MoClo assembly, and are compatible with a number of other common DNA assembly methods.
Building synthetic cells is the main purpose of Nucleus. These are the integrated final products of cytosols, plus content, encapsulated in a container. They can be as simple as liposomes encapsulating water, as complicated as you like, and one day, we hope, as capable as evolved living cells.
Nucleus includes protocols to make three (3) different types of integrated cells.
Getting started? Follow this protocol:
Want more control? Need custom cytosol? Follow this protocol:
Want to implement the detection modules? We are developing the following protocol: ‣. You will make cells capable of detecting and responding to lactose and tetracycline.
Booting up a simple synthetic cell 👋🏾
If you’re new to building synthetic cells, it can be a challenge to learn multiple new protocols that must interoperate. We recommend practicing these protocols by building the “simplest” synthetic cell.
The simplest cytosol to start working with with is the PURE system. PURE is a defined set of proteins, RNA, and small molecules that together can transcribe DNA into RNA and translate RNA into proteins. Multiple commercial implementations of PURE are available, but we recommend PURExpress.
The simplest DNA module to start working with is a fluorescent reporter, like amajLime (included in the Nucleus DNA collection). Whatever module you use, make sure that it has an appropriate promoter for the cytosol that you’ve chosen. PURE expresses proteins under the T7 RNA Polymerase promoter (pT7).
The simplest encapsulation method involves phase-transfer with POPC lipids (mixed with rhodamine tagged lipids for visualization). Once you have all the materials, implementing your first synthetic cell should take a day. A timelapse video of simple synthetic cells is shown on the right (40x magnification).
Developing with synthetic cells 🛠️
For full-stack development with synthetic cells, you’ll want to:
- Build defined cytoplasm directly from individual proteins, RNA, and small molecules. Nucleus includes DNA encoding the thirty-six proteins composing the PURE system. Information for getting started can be found here: .PURE Documentation
- Develop and test your own modules. Nucleus v0.1.0 includes a range of DNA module reporters, including validation modules and detector modules capable of simple logic. It also includes a set of level-matched T7 promoters for expression control. Information can be found here: .
- Encapsulate cytosol and modules. Nucleus v0.1.0 supports encapsulation with the phase transfer method & fluorescent microscope based imaging, and have developed automated analysis methods. Get started here: andContainer ProtocolsContainers Analysis Tools
Check out the