BioBrick

BioBrick parts are DNA sequences which conform to a restriction-enzyme assembly standard. These building blocks are used to design and assemble larger synthetic biological circuits from individual parts and combinations of parts with defined functions, which would then be incorporated into living cells such as Escherichia coli cells to construct new biological systems. Examples of BioBrick parts include promoters, ribosomal binding sites (RBS), coding sequences and terminators. BioBrick parts are DNA sequences which conform to a restriction-enzyme assembly standard. These building blocks are used to design and assemble larger synthetic biological circuits from individual parts and combinations of parts with defined functions, which would then be incorporated into living cells such as Escherichia coli cells to construct new biological systems. Examples of BioBrick parts include promoters, ribosomal binding sites (RBS), coding sequences and terminators. The BioBrick parts are used by applying engineering principles of abstraction and modularization. BioBrick parts form the base of the hierarchical system on which synthetic biology is based. There are three levels to the hierarchy: The development of standardized biological parts allows for the rapid assembly of sequences. The ability to test individual parts and devices to be independently tested and characterized also improves the reliability of higher-order systems. The first attempt to create a list of standard biological parts was in 1996, by Rebatchouk et al. This team introduced a cloning strategy for the assembly of short DNA fragments. However, this early attempt was not widely recognised by the scientific research community at the time. In 1999, Arkin and Endy realized that the heterogeneous elements that made up a genetic circuit were lacking standards, so they proposed a list of standard biological parts. BioBricks were described and introduced by Tom Knight at MIT in 2003. Since then, various research groups have utilized the BioBrick standard parts to engineer novel biological devices and systems. The BioBricks Foundation was formed in 2006 by engineers and scientists alike as a not-for-profit organization to standardize biological parts across the field. The Foundation focuses on improving in areas of Technology, Law, Education and the Global Community as they apply to synthetic biology. BioBricks Foundation's activities include hosting SBx.0 Conferences, technical and educational programs. The SBx.0 conferences are international conferences on synthetic biology hosted across the world. Technical programs are aimed at the production of a series of standard biological parts, and their education expansion is creating acts which help create open, standardized sources of biological parts. As an alternative to traditional biotechnology patent systems and in an effort to allow BioBricks to be utilized as an open-source community standard, the BioBricks Foundation created the BioBricks Public Agreement (BPA). The BPA allows users to establish invention of uses of parts, to disclose patents on parts combinations, and to freely build on the contributions of other users. The BioBrick assembly standard was introduced to overcome the lack of standardization posed by traditional molecular cloning methods. The BioBrick assembly standard is a more reliable approach for combining parts to form larger composites. The assembly standard enables two groups of synthetic biologists in different parts of the world to re-use a BioBrick part without going through the whole cycle of design and manipulation. This means the newly designed part can be used by other teams of researchers more easily. Besides that, when compared to the old-fashioned ad hoc cloning method, the assembly standard process is faster and promotes automation. The BioBrick assembly standard 10 was the first assembly standard to be introduced. Over the years, several other assembly standards, such as the Biofusion standard and Freiburg standard have been developed. Assembly standard 10 was developed by Tom Knight, and is the most widely used assembly standard. It involves the use of restriction enzymes. 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 was the introduction of standard sequences, the prefix and suffix sequences, which flank the 5′ and 3′ ends of the DNA part respectively. These standard sequences encode specific restriction enzyme sites. The prefix sequence encodes EcoRI (E) and Xbal (X) sites, while the suffix sequence encodes SpeI (S) and PstI (P) sites. The prefix and the suffix are not considered part of the BioBrick part. To facilitate the assembly process, the BioBrick part itself must not contain any of these restriction sites. During the assembly of two different parts, one of the plasmids is digested with EcoRI and SpeI. The plasmid carrying the other BioBrick part is digested with EcoRI and Xbal. This leaves both plasmids with 4 base pair (bp) overhangs at the 5’ and 3’ ends. The EcoRI sites will ligate since they are complementary to each other. 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 an 8 base pair ‘scar’ site between the two BioBrick parts. Since the scar site is a hybrid of the Xbal and SpeI sites, it is not recognized by either restriction enzyme. The prefix and suffix sequences remain unchanged by this digestion and ligation process, which allows for subsequent assembly steps with more BioBrick parts. This assembly is an idempotent process: multiple applications don't change the end product, and maintain 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 does not allow the creation of a fusion protein. The scar site causes a frame shift which prevents the continuous reading of codons, which is required for the formation of fusion protein.