BioBrick

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.[7] 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.[8]

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.[9][10]

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

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 as a vector.[12] 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.[13] 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.[3] 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.[13] 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 do not 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.[12] The scar site causes a frame shift which prevents the continuous reading of codons, which is required for the formation of fusion protein.

Tom Knight later developed the BB-2 assembly standard in 2008 to address problems with joining the scars of protein domains and that the scars consist of eight bases, which will yield an altered reading frame when joining protein domains. The enzymes used for digestion of the initial parts are almost the same, but with modified prefixes and suffixes.[14]

The BglBrick assembly standard was proposed by J. Christopher Anderson, John E. Dueber, Mariana Leguia, Gabriel C. Wu, Jonathan C. Goler, Adam P. Arkin, and Jay D. Keasling in September 2009 as a standard very similar in concept to BioBrick, but enabling the generation of fusion proteins without altering the reading frame or introducing stop codons and while creating a relatively neutral amino acid linker scar (GlySer). A BglBrick part is as a DNA sequence flanked by 5′ EcoRI and BglII sites (GAATTCaaaAGATCT) and 3′ BamHI and XhoI sites (GGATCCaaaCTCGAG), and lacking in these same restriction sites internally. The upstream part in the pairwise assembly is purified from an EcoRI/BamHI digest, and the downstream part+vector is purified from an EcoRI/BglII digest. Ligation of these two fragments creates a composite part reforming the original flanking sites required in the part definition and leaving a GGATCT scar sequence at the junction of the parts, a scar that encodes the amino acids glycine and serine when fusing CDS parts together in-frame, convenient due to the GlySer dipeptide being a popular linker of protein domains.[15]

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, and 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 6 bp scar sequence. The 6 bp sequence allows the reading frame to be maintained. The scar sequence codes for the amino acid threonine (ACT) and arginine (AGA).[16] This minor improvement allows for the formation of in-frame fusion protein. However, arginine's being a large, charged amino acid is a disadvantage to the Biofusion assembly technique: these properties of arginine result in the destabilisation of the protein by the N-end rule.

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 sequences 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. The Freiburg standard still forms a 6 bp scar site, but the scar sequence (ACCGGC) now codes for threonine and glycine respectively. This scar sequence results in a much more stable protein[17] as the glycine forms a stable N-terminal, unlike the arginine, which signals for N-terminal degradation. The assembly technique proposed by the Freiburg team diminishes the limitations of the Biofusion standard.

The 3A assembly method is the most commonly used, as its compatible with assembly Standard 10, Silver standard as well as the Freiburg standard. This assembly method involves two BioBrick parts and a destination plasmid. The destination plasmid contains the toxic (lethal) gene, to ease the selection of correctly assembled plasmid. The destination plasmids also have different antibiotic resistance genes than the plasmids carrying the BioBrick parts. All three plasmids are digested with appropriate restriction enzyme and then allowed to ligate. Only the correctly assembled part will produce a viable composite part contained in the destination plasmid. This allows a good selection as only the correctly assembled BioBrick parts survives.

The amplified insert assembly method does not depend on prefix and suffix sequences, allowing to be used in combination with a majority of assembly standards. It also has a higher transformation rate than 3A assembly, and it does not require the involved plasmids to have different antibiotic resistance genes. This method reduces noise from uncut plasmids by amplifying a desired insert using PCR prior to digestion and treating the mixture with the restriction enzyme DpnI, which digests methylated DNA like plasmids. Eliminating the template plasmids with DpnI leaves only the insert to be amplified by PCR. To decrease the possibility of creating plasmids with unwanted combinations of insert and backbone, the backbone can be treated with phosphatase to prevent its religation.[14]

The Gibson scarless assembly method allows the joining of multiple BioBricks simultaneously. This method requires the desired sequences to have an overlap of 20 to 150 bps. Because BioBricks do not have this overlap, this method requires PCR primers to create overhangs between adjacent BioBricks. T5 exonuclease attacks the 5' ends of sequences, creating single-stranded DNA in the ends of all sequences where the different components are designed to anneal. DNA polymerase then adds DNA parts to gaps in the anneal components, and a Taq ligase can seal the final strands.[14]

The 4R/2M assembly method was designed to combine parts (BioBrick Assembly Standard 10 or Silver Standard) within existing plasmids (i.e. without PCR or subcloning). The plasmids are reacted in vivo with sequence-specific DNA methyltransferases, so that each is modified and protected from one of two restriction endonucleases that are later used to linearize undesired circular ligation products. [18]