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Lego bricks are used, mostly by children, to construct vehicles, buildings, and even working robots. Surely the idea of bringing Lego to the lab is childish, but think just for a second… since the human genome has been fully characterized, can we use a Lego-like approach to “build” entire living organisms from scratch?
This may be a very scary thought, but we might be closer than we think! In 2003, Tom Knight at MIT introduced so called BioBrick parts. The BioBricks project aims to provide a workable approach to nanotechnology employing biological organisms. The long-term goal is to produce a synthetic living organism from standard parts that are completely understood.
What are BioBrick parts?
By now you are probably wondering: What are BioBrick parts????? Let me explain:
BioBrick parts are DNA sequences that have a defined structure and function. These parts share a common interface and are designed to be composed and incorporated into living cells to construct new biological systems.
From a practical point of view, BioBrick parts can be incorporated into a circular plasmid in which the functional part is flanked by universal and precisely defined upstream and downstream sequences, called prefix and suffix, respectively. Importantly, these sequences are not considered part of the BioBrick part.
The prefix and suffix contain six restriction sites for specific restriction enzymes. At least two of these restriction enzymes are isocaudomers: that is, pairs of restriction enzymes that have slightly different recognition sequences but upon cleavage generate identical ends. In this way, larger BioBrick parts are created by connecting smaller ones in any desired order. It’s like building a tower with Lego bricks.
There are three levels of complexity using BioBrick parts: parts, devices and systems.
– Parts are the actual building blocks that encode basic biological functions (such as encoding a certain protein, or providing a promoter to let RNA polymerase bind and initiate transcription of downstream sequences).
– Devices consist of multiple parts that implement some human-defined function (such as a riboregulator producing a fluorescent protein whenever the environment contains a certain chemical).
– Systems perform high-level tasks (such as oscillating between two colours at a predefined frequency).
How are BioBricks assembled?
BioBrick parts can be assembled through a process often referred to as ‘Standard Assembly’. This includes normal cloning techniques based on restriction enzymes, purification, ligation, and transformation. When chaining parts together, the restriction sites between the two parts must be removed, allowing the use of those restriction enzymes without breaking the new and larger BioBrick apart. Needless to say, in order to facilitate this assembly process, the BioBrick part itself may not contain any of these restriction sites.
Can I produce my own BioBrick?
Nowadays, a registry of over six thousand public domain BioBrick parts is maintained by Randy Rettberg’s team at http://partsregistry.org. But… what if the BioBrick part you desperately need does not exist??
No need to worry, you can design your own BioBrick in three simple steps:
- Find the sequence of your gene of interest! Tip: use the NCBI genbank.
- Check your DNA sequence for the presence of BioBrick restriction sites. Even if you find them, with a little engineering, you will still be able to create your specific BioBrick. If your sequence is small enough, site-directed mutagenesis can be used to change DNA sequences without changing the protein coding sequence. If the sequence is rather large (thousands of kb), you can use lambda red to alter the DNA sequence.
- And finally, the BioBrick prefix and suffix needs to be added. If your gene is small enough, you can order primers with the BioBricks end sequences and use PCR to generate the desired BioBrick part. If your gene is too large for regular PCR, it is recommended to get your gene synthesized, but be forewarned, this can be a costly process!
Et voila: Your specific DNA sequence with BioBrick ends is ready to be ligated into a plasmid vector that can then be transformed into a suitable host cell.