CH391L/S13/BioBricksAndRegistry

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==History==
==History==
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The registry is an effort that was founded by Tom Knight of the Artificial Intelligence Lab at MIT in 2003. He coined the term "BioBrick" in his paper, "Idempotent Vector Design for Standard Assembly of Biobricks". Idempotent, a term borrowed from mathematics and computer science, in this context means that, during the assembly of complex biological components, the chemical reactions should not alter the key structural elements of the components.<cite>Knight2003</cite>
+
The registry is an effort that was founded by Tom Knight of the Artificial Intelligence Lab at MIT in 2003. He coined the term "BioBrick" in his paper, "Idempotent Vector Design for Standard Assembly of Biobricks". Idempotent, a term borrowed from mathematics and computer science, in this context means that, during the assembly of complex biological components, the chemical reactions should not alter the key structural elements of the components<cite>Knight2003</cite>.
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In the summer of 2004, the registry contained about 100 basic parts; today, this has expanded to over 700 available and 2000 defined parts.<cite>iGEMRegistry</cite>
+
In the summer of 2004, the registry contained about 100 basic parts; today, this has expanded to over 700 available and 2000 defined parts<cite>iGEMRegistry</cite>.
==PoPS (Polymerase per Second)==
==PoPS (Polymerase per Second)==

Revision as of 17:40, 15 April 2013

Contents

Overview of BioBricks and the Registry of Standard Biological Parts

Synthetic biology stands to reap the same benefits from standardization as those that came from standardization in mechanical engineering, like the defining of pitch, diameter, and form of screw threads in the middle of the 19th century.[1]
Synthetic biology stands to reap the same benefits from standardization as those that came from standardization in mechanical engineering, like the defining of pitch, diameter, and form of screw threads in the middle of the 19th century.[1]

The Registry of Standard Biological Parts is a growing bank of genetic building blocks (promoters, DNA binding sites, protein-coding sequences, etc) that are built with the intention of being pieced together to create synthetic systems within organisms. The goal is to create a large functional group of parts (called BioBricks"™), categorized by type, so that new combinations can be built according to the engineering principles of "abstraction" and "standardization".

The principles of abstraction and standardization are what allow engineers to collect, refine, and repackage nature so it's easier to make new and reliable things[2]. Proponents of synthetic biology attest that these principles were never truly integrated into synthetic biology's precursor, genetic engineering. Thus, according to Tom Knight of MIT, who coined the term "synthetic biology", BioBricks and the Registry were created to provide biology with the same advantages similar to those which accompanied standardization in mechanical design - "the widespread ability to interchange parts, to assemble sub-components, to outsource assembly to others, and to rely extensively on previously manufactured components"[1].

BioBricks™ are trademarked in order to define and ensure that the standardized parts in the Registry remain free-for-use as part of a standard library or parts.

"Get Some, Give Some"

The registry is built on the idea of "get some, give some:" the Registry is a resource for users to find and integrate new parts into their systems, while they in turn provide the Registry with data regarding the effectiveness of obtained parts and new parts they have developed. In this way, the Registry continually grows and improves as a community resource.

BioBrick™ Assembly Standard

An actual circular diagram of a BioBrick, flanked by restriction sites and packaged within a plasmid "backbone"[1].
An actual circular diagram of a BioBrick, flanked by restriction sites and packaged within a plasmid "backbone"[1].

A BioBrick is a sequence of DNA with a predefined structure and function. This "payload" is held in a circular plasmid, which is an isolated, circular piece of DNA that can replicate in bacteria.

BioBricks™ are created with the intention of being easily joined and manipulated. In order for this to be possible, the BioBrick™ assembly standard requires the use of defined prefix and suffix sequences (flanking both sides of the BioBrick) that contain specific restriction endonuclease sites. These sites are called EcoRI, NotI and XbaI in the upstream, and SpeI, NotI, and PstI in the downstream. Naturally, the parts must also be engineered such that these sites are not present in the functional region of the sequence[3].

Cutting the BioBrick at specific restriction sites (using restriction enzymes) is what gives a BioBrick its interlocking ends. The end of one BioBrick can then be connected, or ligated, together with the end of another BioBrick, allowing you to effectively string together BioBricks end to end to make devices, and then string devices together to make systems.

For example, to join together two BioBricks, you would first cut both plasmids with restriction enzymes, turning one into an "insert" by getting rid of the rest of the plasmid, and turning the other into a "vector" by opening a space in the plasmid in front of the BioBrick. You then mix together the insert and vector with a special enzyme called a "ligase" that can join together two broken pieces of DNA. Because A's always pair with T's and G's always pair with C's, the overhanging edges of single-stranded DNA that your restriction enzymes left behind will match up to make double stranded DNA. The result is a composite plasmid that contains two BioBricks, now side by side[4].

It is important to note that this new larger composite part has the same restriction sites as the smaller parts it was originally made from. This is what is meant by preserving "key structural elements" that allow one component of any size to be easily connected to any other component[1]. Also note that the "scar" (point of ligation between BioBricks) doesn't match the restriction sites anymore, so the bond between BioBricks will hold through though subsequent rounds of splicing.

Composite components are always created this way, either by “prefixing” one component with another, or “postfixing” one component with another. In both cases, the result is a new, composite component, which can then be used in the same way, as either an insert or a vector, in more complex reactions[1].

A tutorial on BioBrick™ assembly is available on the BioBricks construction tutorial page. Specific assembly standards for different types of BioBricks can be found at the Help:BioBrick Assembly page.

The BioBrick assembly standard was first introduced by Tom Knight in 2003, and he has updated it several times since. The latest draft, from 2008, is called BB2: Draft Standard for Biobrick BB-2 Biological Parts. The standard is still required to be used in all iGEM competitions (as of 2013).

Sharing BioBricks

Restriction enzyme cloning, or "subcloning", is a common way to share BioBrick parts.
Restriction enzyme cloning, or "subcloning", is a common way to share BioBrick parts.

Molecular parts are shared using one of several cloning techniques. One of these techniques is called restriction enzyme cloning, or "subcloning".

Restriction enzymes (or restriction endonucleases) are proteins that cut DNA at or near specific sites. These sites are recognized as a specific DNA sequence, and go by names such as EcoRI, XbaI, SpeI, PstI and NotI. Assuming the your gene of interest (YGOI for short) exists in a bacterial plasmid or vector (donor plasmid), the restriction enzymes are used to cut YGOI out of the donor plasmid and then cut the recipient plasmid at a specific location in a specific pattern, so that YGOI can then be "pasted" to that location in the recipient plasmid using a process called ligation[5] [6].

History

The registry is an effort that was founded by Tom Knight of the Artificial Intelligence Lab at MIT in 2003. He coined the term "BioBrick" in his paper, "Idempotent Vector Design for Standard Assembly of Biobricks". Idempotent, a term borrowed from mathematics and computer science, in this context means that, during the assembly of complex biological components, the chemical reactions should not alter the key structural elements of the components[1].

In the summer of 2004, the registry contained about 100 basic parts; today, this has expanded to over 700 available and 2000 defined parts[7].

PoPS (Polymerase per Second)

MIT initially used a unit of measurement called TIPS (Transcription Initiations per Second) for measure rates of transcription at the ends of its parts; however, this was insufficient because there are places on the DNA (e.g. terminators) where transcription initiations are not taking place. PoPS is a relatively new unit developed during construction of standardized "ends" of DNA pieces that measures the inputs and outputs of BioBrick™ parts. PoPS measure the rate at which RNA polymerase moves past a point in the DNA, similar to measuring the current flow across a specific point in a wire. Devices that have an input and output in PoPS are composable - that is, they can be arbitrarily joined together to create complex devices and systems. Creation of devices allows us to characterize devices and eventually more complex systems, thus PoPS is important as a common signal carrier. PoPS differs from transcription rate in that it can also be measured at terminator sites; upstream, they are theoretically equivalent.

An example of a system from which PoPS is understandable is a PoPS based inverter, which takes in a PoPS signal and inverts it. A PoPS based inverter consists of a ribosome-binding site, repressor coding region, terminator and cognate promoter. A high PoPS input cause expression of the repressor, which then binds to the promoter and produces a low output signal. A low PoPS input means very little repressor expression, so the promoter is free to generate PoPS.

At the end of the day PoPS is a useful abstraction that we can use to think about transcription-based logic devices and characterize BioBrick™ parts; up to this point, there has been to way of measuring it in vivo.[8]

Using the Registry

Main Page

The main page of the registry has a welcome message, four main icons, a list of registry tools, and registry news. The areas of interest for the purposes of searching and finding parts and the iGEM competition are the main tabs and registry tools.

Icons
This tab opens up the Catalog of parts, devices, and systems for browsing and finding parts.
  • Help
The help section of the page consists of FAQ's, an introduction to BioBrick standardized parts, instructions for various types of assembly, information about the Registry and its tools, a more in-depth look at designing systems using BioBricks©, information about the DNA Repositories, and help for users and groups.
  • Users & groups
This icon links to the iGEM competition home page.
  • DNA repositories
This icon opens up the DNA Part Repositories page, which contains a list of (and a convenient search feature for) the DNA for BioBricks available in plasmids in cells.
Registry Tools
Opens the search page for parts in the Registry.
  • Add a part
Opens the page for adding basic parts, composite parts, and construction intermediates to the Registry.
Opens the page where parts can be requested for use by iGEM teams.
  • Opens the instructions for preparing and sending DNA to the Registry.
  • Allows you to use quick analysis of a single sequence or begin a more complex sequencing project in order to compare a sequence to the parts or a specific part in the Registry, combine several sequence readings to see if your part is correct, or save a sequence with the part's other information so future users can find it.

Catalog

The registry is focused primarily around the Catalog containing sorted and categorized entries. The Catalog is split into a hierarchy of parts, devices, and systems. Parts are the simplest entries in the catalog, basic building blocks for devices and systems. Devices consist of multiple parts pieced together to perform a particular function. Systems, the most complex of the three, are self-contained sequences that entirely specify all the parts encoding a device designed for a specific task.[9]

You can browse the parts and devices in the Catalog by:

  • Type
  • Function
  • Chassis (the model in which the part works best)
  • Assembly standard (each assembly standard is described in detail with the correct parts and methods included on the catalog page for that method)
  • Contributor
  • Other user-generated catalog pages

BioBrick™ Types

Screenshot of BioBrick types within the Registry catalog.
Screenshot of BioBrick types within the Registry catalog.

At a high level, the main types of BioBrick parts are "protein coding sequences", "promoters", and "primers". Protein coding sequences are like “recipes” that are read by a chemical called “polymerase” and then transcribed into RNA which is then translated into proteins like... bioluminescence, banana smell, and colors.

  • Protein coding sequences are like “recipes” that are read by a chemical called “polymerase” and then transcribed into RNA which is then translated into proteins like... bioluminescence, banana smell, and colors.
  • A promoter is a “switch” upstream of a coding sequence that controls when a protein actually gets made. In other words, it controls when the polymerase begins transcription into RNA. The frequency at which a protein is made can be measured, and the current standard of measurement is called "PoPS", or "polymerases per second".
  • A primer allows you to select a specific region of DNA in order to make copies of it (called “amplification” or “cloning”).

BioBrick™ Names

The letters used in naming parts and their corresponding functions.
The letters used in naming parts and their corresponding functions.

The names of parts in the Registry begin with BBa = BioBrick [version] alpha, followed by a letter indicating their function. These letters and their corresponding functions are displayed in the image to the right.

An example of a part is BBa_I721001. The easiest way to determine the function of this part is simply to take the name and enter it into the search bar in the top right corner of the page. I attempted searching multiple parts based on their name in the actual search page of the registry, however it could not find them. It is possible that this is due to them recently redoing their system, because the part has been in the registry since 2007 in this case. Decoding the name is fairly simple; the first part implies that it is a BioBrick part type alpha (I can't find any examples of beta parts in the system). The I, as shown in the chart to the right, is supposed to signify that it is from an IAP project from 2003 or 2004; however, this is not the case (explained below). The numbers are assigned to groups involved in the project; searching the part will pull up its page with that information. In this case, the part was contributed by Jeffrey Hoffman and his iGEM group from 2007 - this is a clear example of the mentions in the paper by Peccoud [10] that a large percentage of parts in the registry are mislabeled or inaccurate.

Finding a Part

The easiest way to find a basic part if you know the part name or number is to enter the information on the search page of the Registry. If you are searching for parts that serve a specific purpose, the quickest way is to browse the parts by type or function in the Catalog. Finding composite parts entails the same process as basic parts; to find composite parts that contain a specific basic part you can use the Superpart search section on the Registry search page.

This information and more can be found in greater detail on the registry's Help:Search page.

Ordering Parts

Once you've found a part you want to order, there are multiple ways of acquiring it from the registry. Before ordering, you should go to the part's main page on the registry and check if it's available. This information is in a box on the top right of the page, listing the DNA as available if the part can be requested. Once you've checked availability, it can be requested via email. An email should be sent to hq@igem.org containing your iGEM team or lab name, the name of the part, the plasmid backbone and resistance, and the source plate and well. This information and more is on the Registry's site on the Help:Requesting Parts page.

Addgene

Addgene is a nonprofit organization whose purpose is to create a plasmid repository that will ease sharing of plasmids between scientists. Plasmids can be found in the following categories: empty backbone, species of gene, popular plasmids, depositing scientist, special collections, expression system, consortiums, and vector type. Plasmids can be ordered directly from their website. Several useful tools are available on their website as well, including a sequence analysis program, vector database, and various protocols for operations involving plasmids.

Legacy

2013 was the ten-year anniversary of the creation of the BioBrick standard, the Registry of Standard Biological Parts, and the iGEM competition. Technology has advanced to the point that it is can be more practical to encode and manufacture a complex DNA sequence locally and directly, rather than order BioBricks and splice them together one at a time.

However, BioBricks and the Registry are still in use today by many iGEM teams that don’t have access to the latest technology and equipment.

Here is an informational video about synthetic biology, published in 2011, that references the registry: "Synthetic Biology Explained" by James Hutson, Bridge8

References

  1. Knight, Tom. Idempotent vector design for standard assembly of biobricks. MASSACHUSETTS INST OF TECH CAMBRIDGE ARTIFICIAL INTELLIGENCE LAB, 2003. [Knight2003]
  2. "Synthetic Biology Explained" <http://www.youtube.com/watch?v=rD5uNAMbDaQ> [SynBioExplained]
  3. Canton B, Labno A, and Endy D. . pmid:18612302. PubMed HubMed [Canton2008]
  4. http://agapakis.com/hssp/biobricks.html [agapakis]
  5. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Cloning: A laboratory manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 1.63-1.70. [CloningColdSpringHarbor]
  6. http://homepage.univie.ac.at/nikos.pinotsis/webPP/genetoprotein/cloning_strategy/clo_rest-enzymes.html [CloningNikosPinotsis]
  7. http://partsregistry.org/Help:About_the_Registry [iGEMRegistry]
  8. "PoPS." OpenWetWare, . 4 Oct 2007, 16:12 UTC. 8 Feb 2012, 21:11 <http://openwetware.org/index.php?title=PoPS&oldid=156219>. [PoPS]
  9. Peccoud J, Blauvelt MF, Cai Y, Cooper KL, Crasta O, DeLalla EC, Evans C, Folkerts O, Lyons BM, Mane SP, Shelton R, Sweede MA, and Waldon SA. . pmid:18628824. PubMed HubMed [Peccaud2008]
All Medline abstracts: PubMed HubMed
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