User:Shammie23/sandbox

List of BioBrick parts represented with symbols

BioBrick parts are DNA sequences which have been standardized and conform to the BioBrick assembly standard.These Lego-like buiding blocks are used to design and assemble synthetic biological circuits, which would then be incorporated into living cells such as E-coliE coli to construct new biological systems.Examples of BioBrick parts includes Promoter,Ribosomal binding site (RBS), coding sequence and terminator.

Parts, device and system

Abstraction hierarchy allows the breakdown of complexity.

The BioBrick parts are treated as electrical components as it is standardized and characterised by applying engineering principles of abstraction and modularisation. Biobrick parts forms the base of the hierarchical system of which synthetic biology is based upon. There are 3 levels to the hierarchy: parts, device and system.

• Parts: pieces of DNA that forms a functional unit (example Promoter, RBS, etc.)
• Device: collection sets of part with defined function. In simple terms, a set of complimentary Biobrick parts put together forms a device.
• System: Combination of set of devices that performs high-level tasks.

History

The first attempt to have a list of standard biological part was in the year 1996, by Rebatchouk et al. Rebatchouk and team introduced cloning strategy for the assembly of short sections DNA fragments. However, their attempt fruitless because it wasn't recognized by the scientific research community back then.Not long after that, in the year 1999, Arkin and Endy realized that the heterogeneous elements that make up a genetic circuit were lacking standards and hence proposed a list of standard biological parts , which was also unrecognised of its full potential. Biobrick parts were introduced by Tom Knight at MIT in 2003. Since then, various research groups have utilized and used the BioBrick standard parts to engineer novel biological devices and system.

BioBrick Assembly standard

BioBrick assembly standard was introduced to overcome the lack of standardization posed by the traditional molecular cloning method. BioBrick assembly standard is a more reliable approach to combine BioBrick parts to form a new composite part. An assembly standard enables two groups of synthetic biologists in different parts of the world to re-use a BioBrick part which going through the whole cycle of design and manipulation. This means the newly designed part can be used by other team of researchers as well without a problem in the future. Besides that, when compared to the old fashioned ad hoc cloning method, the assembly standard process is faster and promotes automation. BioBrick assembly standard 10 was the first ever assembly standard to be introduced. Over the years, several other assembly standards, like the Biofusion standard and Freiburg standard was developed.

BioBrick assembly standard 10

Standard assembly of two BioBrick parts(promoter and coding sequence) by digestion and ligation which forms a 'scar' site(M).

DNA assembly 10 was developed by Tom Knight, is the most widely used and accepted assembly standard, which involves restriction enzymes (RE). Every Biobrick part is a DNA sequence which is carried by a circular plasmid, which acts a vector. The vector acts as a transport system to carry the Biobrick parts. The first approach towards a BioBrick standard involves the introduction of standard sequences, the prefix and suffix sequences which flanks the 5’ and 3’ end of the DNA part respectively.These standard sequences encode for specific restriction enzyme restriction sites. The prefix sequence encodes for EcoRI (E) and Xbal (X),while the suffix sequence encodes for SpeI (S) and PstI (P).The prefix and the suffix are not considered part of the BioBrick part. To facilitate this assembly process, the BioBrick part itself will not contain any of these restriction sites. Altogether, a plasmid carrying a DNA part contains four different restriction enzyme recognition sites. During the assembly of two different parts, one of the plasmids is digested with SpeI and Xbal. While the other plasmid carrying the other BioBrick part is digested with EcoRI and Xbal. This leaves both the plasmid with 4 base pair(bp) overhangs in both the 5’ and 3’ end. The EcoRI sites will ligate since they are the same. The Xbal and SpeI sites will also ligate as the digestion produces compatible ends. Now, both the DNA parts are in one plasmid. The ligation produces a 8 base pair ‘scar’ site between the two BioBrick parts. Since the scar site is a hybrid of the X and S site, it is not recognized by either restriction enzymes. The prefix and suffix sequence remains unchanged as before the digestion process, this allows for subsequent assembly steps which would allow more BioBrick part to be combined with the existing one.

This assembly is done is an idempotent fashion, where multiple applications don’t change the end product, refers to the maintenance of the prefix and suffix. Although the biobrick standard assembly allows for the formation of functional modules, there is a limitation to this standard 10 approach. The 8 bp scar site doesn’t allow the creation of a fusion protein. The scar site causes a frame shift which prevents the continuous read of codons, which is required for the formation of fusion protein.

Silver standard

Biofusion assembly of two BioBrick parts.The schematic diagram shows the 6 base pair scar site made due to the deletion and insertion of nucleotide in the XbaI and SpeI sites.

Pam Silver’s lab created the Silver assembly standard to overcome the issue surrounding the formation of fusion protein. This assembly standard is also known as Biofusion standard ,is an improvement of the BioBrick assembly standard 10. Silver’s standard involves deletion of one nucleotide from the Xbal and SpeI site, which shortens the scar site by 2 nucleotides, which now forms a 6bp scar sequence. The 6bp sequence allows the reading frame to be maintained.The scar sequence codes for the amino acid threonine (ACT) and Arginine (AGA). This minor improvement allows for the formation of in-frame fusion protein. Arginine being a charged amino acid as well as its big size lands as a disdvantage to the Biofusion assembly.These properties of arginine restrict the flexibility of the scar site and doesn't comply with the N-end rule.

Freiburg standard

The 2007 Freiburg iGEM team introduced a new assembly standard to overcome the disadvantages of the existing Biofusion standard technique. The Freiburg team created a new set of prefix and suffix sequence by introducing additional restriction enzyme sites, AgeI and NgoMIV to the existing prefix and suffix respectively. These newly introduced restriction enzyme sites are biobrick standard compatible which allows for the creation of a more codon friendly scar site. The Freiburg standard still forms a 6bp scar site, but then instead of Thr-Arg, the scar sequence (ACCGGC) now codes for threonine and glycine respectively. This scar sequence satisfies the N-rule as the glycine forms a stable N-terminal, unlike the N-term Arg, which signals for N-terminal degradation. The assembly technique proposed by the Freiburg team diminishes the limitations of the Biofusion standard.

Assembly method

There are different methods for assembling parts: some are due to different standards requiring different materials and methods (i.e. restriction enzymes), others are due to preferences in protocol (faster assembly, higher efficiency, pcr, ease of use, etc). Method used during the assembly process.

3 Antibiotic (3A) assembly

THe 3A assembly method is comaptible with assembly Standard 10, Silver standard as well as the d Freidburg standard.

Scarless /Gibson assembly

9 There are assembly methods which also allow for the construction of composite parts without scars or specific linkers, which is particularly useful for the assembly of proteins. Additionally, these methods may be able to circumvent assembly standard incompatibility between part samples: a part sample in a Silver RFC[23] plasmid backbone can be assembled with a part sample in a Berkeley RFC[21] plasmid backbone.Gibson Assembly-Gibson Assembly has not been tested by the Registry yet, but several teams have had success with this assembly method. The Cambridge 2010 iGEM Teamdeveloped a set of protocols and tools that may be useful. The Registry will be evaluating Gibson Assembly, and will have resources available for this assembly method available soon.)

Parts Registry

The MIT group lead by Tom Knight that developed BioBricks and International Genetically Engineered Machines (iGEM) competition are also the pioneers of The Registry of Standard Biological Parts (Registry). Registry being on of the foundations of synthetic biology, provides web-based information and data of over 2,000 BioBrick parts. The Registry contains:

• Information and characterisation data for all parts, device and system
• Includes a catalogue which describes the function ,performance and design of each part

Every BioBrick part has its unique identification code which makes the search for the desired BioBrick part easier(for example, BBa_J23100, a constitutive promoter). The registry is an open access domain, whereby anyone can submit a BioBrick part. Most of the BioBrick submission is from students participating in the annual iGEM competition hosted every summer. Renowned scientists’ as well academic labs from all over the world also submit BioBrick parts to the registry. The Registry allows exchange of data and materials online which allows rapid re-use and modifications of parts by the participating community.

Professional parts registries have also been developed due to the limitation imposed by the parts Registry that is high collaborative with iGEM. Since most of the BioBrick parts are submitted by undergraduates as part of the iGEM competition, the parts may lack important characterisation data and metadata which would be essential when it comes to designing and modelling the functional components. One example of a professional parts registry is the USA -based public-funded facility, The International Open Facility Advancing Biotechnology (BIOFAB). Although BIOFAB was just established recently, it contains detailed description of each biological part, it is also an open-source registry which is available commercially. BIOFAB aims to catalogue high-quality BioBrick parts to accommodate the needs of professional Synthetic Biology community. The well-defined BioBricks parts available from BIOFAB would be useful in the design and modelling of novel biological systems.

The BioBrick Foundation (BBF), is an public-benefit organisation established to promote the use of standardized BioBrick parts on a scale beyond the iGEM competition. The BBF is currently working on the derivation of standard framework to promote the production high quality BioBrick parts which would be freely available to everyone.