Difference between revisions of "CH391L/S14/BiologicalpartsandtheiGEMregistry"

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* [http://parts.igem.org/Protein_coding_sequences Coding Sequences] (Exons of genes or other sequences that when translated lead directly to polypeptides)
 
* [http://parts.igem.org/Protein_coding_sequences Coding Sequences] (Exons of genes or other sequences that when translated lead directly to polypeptides)
 
* [http://parts.igem.org/Terminators Terminators] (Genetic structures that prevent transcription from continuing, usually after a gene)
 
* [http://parts.igem.org/Terminators Terminators] (Genetic structures that prevent transcription from continuing, usually after a gene)
 +
[[Image:02022014_main_page_BBaJ45001.png | thumb | right | 450 px | Main page for the [http://parts.igem.org/Part:BBa_J45001 BBa_J45001] BioBrick.  The other pages for the BioBrick can be seen along the top.]]
 
There are also ways to organize by plasmid backbones, chassis, or function.  Each BioBrick has four pages related to it:
 
There are also ways to organize by plasmid backbones, chassis, or function.  Each BioBrick has four pages related to it:
 
* Main Page: Gives a basic overview of how the BioBrick is used and how it functions
 
* Main Page: Gives a basic overview of how the BioBrick is used and how it functions

Revision as of 04:50, 3 February 2014

Contents

Introduction

Biological parts are refined, functional DNA sequences that have been standardized so as to act the same way in any biological device. These DNA sequences may be coding regions from genes, regulatory elements, or other genetic elements.[1] The Registry of Standard Biological Parts, or iGEM Registry, is a collection of these parts, commonly referred to as Biobricks™, that is being expanded constantly and can be used freely to create biological devices. Located at the Massachusetts Institute of Technology, the registry stocks BioBricks and biological devices that have been previously designed and accepts any new BioBricks (that adhere to the specifications) that are submitted.


History

Genetic engineering has grown rapidly since the 1970s with the development of techniques and other advancements in the manipulation of DNA and RNA both in living organisms and out. Biological devices, DNA structures synthesized from various natural sources with a set function that draws on all of its parts, started to emerge but were not very cohesive, which led to difficulties in combining them. In 2003, Tom Knight, a researcher at MIT, developed the idea of the BioBrick, a standardized design for biological parts that can be combined with each other with no complications or unforeseen results.[2] Knight's idea was to apply the principles of mechanical design to genetic engineering – that is, making it so that each part is easily interchangeable without losing functionality. Around the same time at MIT, the International Genetically Engineered Machine (iGEM) competition began, allowing these iGEM teams to create biological devices and compete to see who's device was the best, giving incentive to create these biological parts. As the competition increases each year, the parts and devices from the previous years are stored and available for use, and the number and quality of parts increases. The parts are stored in the Registry of Standard Biological Parts using the BioBrick format, and can be ordered by iGEM teams or synthetic biology labs. The underlying idea behind the Registry and biological parts is being able to combine parts in different ways easily and without unwanted effects, which is possible due to standardization.

Standardization

Standardization is key to the iGEM registry's system. Without the standards set by the registry, the BioBricks would not be able to be combined in different ways and still function the same. The key to standardization for BioBricks is the way each one is assembled: there is a very specific set of parts in each BioBrick which allows them to be combined easily and in any order.[2] An important idea for standardizing the process of making BioBricks is idempotence. Idempotence when used in terms of synthetic biology means that “each reaction leaves the key structural elements of the component the same.”[2] In other words, the BioBricks can be exposed to most reactions and environments without changing its key structure and function. This makes the parts much more reliable and easier to work with. Related to the idea of standardization is abstraction, another key feature of synthetic biology and the iGEM registry.[3] Abstraction refers to the idea of taking things out of context and looking at just the core features and behaviors. With regards to synthetic biology, that means that while the function of each individual DNA sequence may seem very complex, there are ways to categorize DNA sequences to allow for smoother usage and classification of biological parts. Parts and devices can be grouped on different levels of complexity such as the overall category they belong to (e.g. promoter or terminator), the subset of biological function they fall under (like reporter gene or biosynthesis part), or the specific function that they perform. This allows new parts and devices to be categorized more quickly, and knowing what category each part belongs to helps to combine them more efficiently.

Assembly Standards

Aside from the BioBrick standard, many other assembly standards have been introduced, with a few key ones being allowed as an acceptable format for submitted parts in the Registry.

  • BioBrick RFC[10]: The basic BioBrick standard, based on idempotent assembly with the prefix and suffix discussed here. It does not allow for the assembly of proteins, as the scar created by combining two parts contains a stop codon.
  • BioBrick BB-2 RFC[12]: Designed to fix the problems with the BioBrick RFC[10] standard, this one does allow for in-frame protein assembly. it is, however, incompatible with the RFC[10] standard.
  • Berkeley RFC[21]: Optimized to allow for in-frame protein assembly. It used BamHI and BgIII restriction enzymes. It is incompatible with the RFC[10] standard.
  • Silver RFC[23]: Based on the RFC[10] standard, but has a shorter, 6 bp scar that allows for in-frame protein assembly. However, the scar includes an Arg codon, which will degrade the protein faster.
  • Freiburg RFC[25]: An extension of the RFC[10] standard that includes two extra restriction sites to allow for in-frame assembly of protein domains. It has some compatibility with RFC[10].

BioBricks

Standard prefix and suffix of a BioBrick[4]

BioBricks consist of a few set components, plus the specific functional DNA sequence that will be used in other biological devices. Each BioBrick must have a set prefix and suffix: the prefix contains EcoRI, NotI, and XbaI restriction enzyme sites, while the suffix contains SpeI, NotI, and PstI restriction enzyme site. Because the sticky ends left by the XbaI and SpeI restriction sites are complementary, they can bind to each other to form a mixed site where neither restriction enzyme can bind.[4] This means every BioBrick has a common point to bind to the next that will not be broken if exposed to either of the restriction enzymes again. The prefix and suffix are what allow multiple BioBricks to be combined easily. Outside of these parts, the BioBrick also consists of a plasmid backbone with a replication origin and antibiotic resistance marker, two parts necessary for cloning the plasmid (and the BioBrick with it).

Function

Plasmid of the BBa_J45001 BioBrick.

Each BioBrick has one function, and will complete only that function when combined with other BioBricks in a biological device. The function is often derived from the function of the DNA sequence the BioBrick was synthesized from. For instance, BBa_J45001, which encodes for a protein that converts salicylic acid to methyl salicylate (artificial wintergreen oil), is derived from Antirrhinus majus, a plant that naturally produces said protein. The most common way of finding proteins such as this is by looking for the desired function in various organisms and identifying the DNA sequence that causes the function. Then, the BioBrick is created by refining the DNA sequence in such a way that it only includes the desired function, and will not be affected by other biological parts or devices.






iGEM Registry

The iGEM registry collects the BioBricks and biological devices into one catalog from which iGEM teams and synthetic biology labs can order the parts they need. The catalog is organized online by type and function, with the following main categories:

  • Promoters (Increase the expression of a DNA sequence downstream)
  • Ribosome Binding Sites (Increase the binding ability of ribosomes to mRNA)
  • Coding Sequences (Exons of genes or other sequences that when translated lead directly to polypeptides)
  • Terminators (Genetic structures that prevent transcription from continuing, usually after a gene)
Main page for the BBa_J45001 BioBrick. The other pages for the BioBrick can be seen along the top.

There are also ways to organize by plasmid backbones, chassis, or function. Each BioBrick has four pages related to it:

  • Main Page: Gives a basic overview of how the BioBrick is used and how it functions
  • Design: Information about the DNA sequence of the BioBrick and the plasmid, the ability of the BioBrick to be combined with different restriction enzymes, notes about the design process, and the source of the DNA sequence
  • Experience: Allows users to submit their experiences using the BioBrick in their own biological devices
  • Information: Technical information about the BioBrick itself, such as the original designer, part type, status, qualitative experience (whether it has worked in the past or not), and short description of its function

There is also a “part tools” section which includes related parts, the length of the plasmid, and sequence analyses, as well as ways to edit parts of the page. Finally, there is a link to order the part (assuming it is available).[5]

Submitting and Ordering Parts

To submit a part, you first choose whether you are submitting a basic part, a composite part (made of more than one part added together), or a construction intermediate, which has no function and is the (usually unwanted) byproduct of assembling two parts together.

To order a part, go to the page of the part you want to order and click the link that says "order this part". It will take you to a page listing four options: get the part from a Registry distribution, request the part from the Registry, have the part synthesized, or use PCR to standardize an existing sample. The first two are options in which you get the part from the Registry, while the last two involve you making the part yourself. Should you order the part from the Registry, it will arrive as an agar stab, lysogeny broth (LB) agar with bacteria containing the BioBrick plasmid. The bacteria have to be incubated on a plate in order to extract the plasmid from it.


Future Directions

The iGEM Registry is a constantly updating resource, so it requires a lot of upkeep to run smoothly. There are many problems or missing information on the various pages of specific BioBricks, and the website is often undergoing changes to make finding and ordering the parts smoother. Furthermore, the idea that BioBricks are the best method of standardization is being challenged. Many iGEM teams and synthetic biology labs are coming up with ideas to improve on the BioBrick standard and the other standards the iGEM Registry uses. While most of these are not very widespread, they may be more efficient or more useful than the current standards. This line of investigation is definitely something the iGEM Registry should look into. Most recently, the website was redesigned to simplify the page for each part, giving it just five individual pages to work with, while expanding the information available for it.[5] The iGEM Registry will continue to be refined and improved while expanding by the various additions from iGEM teams and synthetic biology labs around the world.


References

  1. Canton, B., Labno, A., and Endy, D. (2008) Refinement and standardization of synthetic biological parts and devices. Nature Biotechnology 26:787-793. [Canton2008]
    Discussion of the purpose and ideas behind designing biological parts and devices.
  2. Knight, T., "Idempotent Vector Design for Standard Assembly of BioBricks" (MIT, Cambridge, MA, 2003). [Knight2003]
    Explanation of how and why BioBricks are designed and used.
  3. Endy, D. (2005) Foundations for engineering biology. Nature 438:449-453. [Endy2005]
    Introduction and overview of the principles and ideas of synthetic biology.
  4. Ho-Shing, O., Lau, K., Vernon, W., Eckdahl, T., Cambell, A. (2012) Assembly of standardized DNA parts using BioBrick ends in E. coli. Methods in Molecular Biology 852:61-76. [Ho-Shing2012]
    General methods and basic usage of BioBricks.
  5. Registry of Standard Biological Parts. [1] [Registry]
    Online catalog of standard biological parts. Can be updated with new biological parts that fit the criteria.