By Jin Wang, Ph.D.
CAS Key Laboratory of Synthetic Biology
Institute of Plant Physiology and Ecology
Shanghai Institutes for Biological Sciences
Chinese Academy of Sciences, Shanghai 20032, China
The BioBricksTM standard was originally introduced by Knight et al. (http://hdl.handle.net/1721.1/21168) in 2003. Since its birth, many efforts have been made to improve the original standard (called BBF RFC 10) by participants around the world. Indeed, so far, over 100 BBF RFC schemes have been developed and more than 20,000 BioBricks™ parts have been constructed. More importantly, more and more scientists have recognized the significance of the BioBricksTM standard and are willing to build and use BioBricksTM parts in their research work.
In this group, our study has been focused on elucidating the molecular mechanisms of regulation for secondary metabolites in actinomycetes. In particular, our group found decades ago, that addition of nitrate into the fermentation medium remarkably stimulates the yield of rifamycin in the bacterium Amycolatopsis mediterranei. This finding has been widely used in improving industrial fermentation ever since, and is called the “nitrate stimulating effect” . Subsequent physiological and biochemical studies revealed the pleotropic characteristics of this effect, and systematic studies have been carried out to elucidate the molecular mechanisms underlining this “global regulation.” Along with that work, whole genome sequences of Amycolatopsis species were generated , and hundreds of different kinds of biosynthetic clusters have now been annotated for potentially useful production of secondary metabolites.
Eventually, we would like to clone these clusters in the form of “biological parts” to facilitate further studies, e.g. in combinatorial biosynthesis and other fields. However, it is inconvenient to use the present BioBrickTM standards for manipulation of these large clusters or genes because there are a large number of restriction sites within the DNA sequences. Because these sites must be removed before they can be used in building the bricks, the use of these pieces will undoubtedly cost huge amounts of time and money. In fact, a lot of scientists have been troubled by the procedure of removing massive restriction sites. With these considerations in mind, we wondered whether we could invent some adaptors with long recognition sequences, sparsely distributed in natural DNA sequences. Luckily, we have found that a pair of homing endonucleases, I-SceI and PI-PspI, recognize long DNA sequences, and both produce the cohesive termini of “TTAT.” These endonucleases are therefore suitable for the establishment of a new standard for bricks construction, as we described in our recent paper in PLOS ONE .
We would like to give an attractive name to this standard in order to distinguish it from others. As some homing endonucleases can tolerate a small number of basepair substitutions within their targets without a loss in enzyme efficiency, we first chose I-CeuI, which produces an end sequence of “TTAG,” and tried to mutate the recognition sequences of the two enzymes to produce the same termini. Because the names of both intron-encoded endonucleases contain “i”, we designated the new standard as iBrick. Then, the I-CeuI target was mutated to produce the “TTAT” terminus, which was the same as that of I-SceI, but we found the mutated sequence was poorly recognized and digested by I-CeuI, and vice versa. Luckily, we then found that PI-PspI could directly produce “TTAT,” and so we used it instead. However, we maintained the name of the standard, despite this change. In addition, “i” has the same pronunciation as love (“ai”) in Chinese. Therefore, “iBrick” is similar to “love brick,” and we hope synthetic biologists, particularly those working on large and complex bricks, will love the idea of iBrick.
Similar to the present BioBricksTM standards, e.g. BBF RFC 10, iBrick uses simple cut-ligation procedures to make any desired constructs. However, as the recognition sites are extremely rare among the natural DNA sequences, the iBrick standard is convenient for direct assembly of large DNA pieces. In the present version (iBrick version 1.0), a 21-bp scar is produced between bricks. Although the 21-bp sequence contains no stop codon, it is still much longer than the present BioBricks and BglBrick standards, which usually contain a 6-bp sequence. After communicating with many synthetic biologists, we do understand that this long scar might be the primary obstacle for us to promote the iBrick standard. In addition, as pointed out by Professor Barry Stoddard, within the seven-amino acid sequence there are three leucine residues, which are hydrophobic and may cause problems with protein solubility when employed in protein fusion applications. Therefore, improvements to the iBrick standard are now ongoing, mainly focusing on reducing the length of the scar produced and optimizing the identity of amino acids within the scar. One such attempt involves using the TALEN technology to create new enzymes that specifically bind to the recognition sequences of I-SceI and PI-PspI, but cut outside of them to remove excess prefixes and suffixes, thus producing short scars after ligation.
Ultimately, we do hope the improved iBrick standard that produces a short scar can be widely used by synthetic biologists all around the world.
1. Jiao, R.S., et al., Studies on the metabolic regulation of biosynthesis of rifamycin by Norcadia (Amycolatopsis) mediterranei I. The stimulative effect of nitrate on biosynthesis of rifamycin SV by Nocardia mediterranei. Acta Phytophysiol Sinica, 1979. 5: p. 395-402.
2. Zhao, W., et al., Complete genome sequence of the rifamycin SV-producing Amycolatopsis mediterranei U32 revealed its genetic characteristics in phylogeny and metabolism. Cell Res, 2010. 20(10): p. 1096-108.
3. Liu, J.K., et al., iBrick: A New Standard for Iterative Assembly of Biological Parts with Homing Endonucleases. PLoS One, 2014. 9(10): p. e110852.