New user-friendly DNA assembly toolkit
The development of a more versatile, simple and efficient DNA assembly kit will offer researchers unlimited ways of building complex molecular constructs by combining different DNA parts.
The ability to assemble different DNA parts is essential to synthetic biology, which enables researchers to construct new biological pathways or redesign existing biological systems.
Existing DNA assembly toolkits typically compromise on either simplicity, the complexity of the assembly process, or capacity, the number of DNA parts that can be combined.
Researchers from the University of Edinburgh have tackled those issues by creating Mobius Assembly, which combines unlimited assembly capacity in a simple, streamlined process.
Synthetic biology uses engineering principles on a nanoscale, to assemble different DNA parts into longer stretches of DNA that result in new cell functions, and sometimes to create entire biological pathways.
Mobius Assembly uses a two-stage design that allows a complex DNA construct to be made by combining a series of smaller DNA parts – similar to building a computer.
In one step researchers pick from a library of standardized DNA parts, to build a functional unit, similar to combining the right parts together to make a USB port or the circuit board.
In the second stage, these larger functional units are joined together to make a system – rather like bringing all the bigger parts together to make a working laptop.
Mobius Assembly allows researchers to switch back and forth between these stages, called levels, so that new parts can be continually added to the system, allowing a complex DNA sequence to be assembled.
Identifying Successful DNA Additions
Mobius Assembly users can also visually distinguish between the different assembly levels and identify successful DNA additions by the use of coloured chromogenic proteins.
The chromoproteins are produced by bacteria, eliminating the need for researchers to add toxic and expensive chemicals to screen for successful DNA assembly.
Understanding through Design
By breaking genomes down to their individual parts researchers gain a better understanding of how living systems work.
Synthetic biology also allows the design of new biological processes, such as developing drug-producing bacteria in which a whole new biosynthetic pathway has been introduced.
Living cells, such as yeast or bacteria, act as a ‘blank canvas’ which house the new, synthetic DNA constructs – effectively turning them into biofactories.
Assembling even simple DNA constructs is more complex than building a computer. The way the DNA parts interact is complex and sometimes unknown - making it similar to working in black box.
The most common method for DNA assembly uses enzymes, called endonucleases, to cut out the DNA parts at specific sites leaving sticky ends, or overhangs.
These overhangs pair to other complementary ones on other DNA parts, allowing different DNA parts to combine in a specific order.
To improve its versatility, the researchers built the Mobius Assembly toolkit using sticky overhangs most commonly used by researchers allowing its parts to be shared widely.
The researchers also introduced a rare-cutter endonuclease, which is more specific and ensures the DNA parts can be extracted intact with less modifications needed - speeding up the assembly process.
“We made Mobius Assembly as user-friendly as possible. We currently use it with Escherichia coli and plant cells, but we hope it will be used widely and adapted to many different types of organisms.”
Mobius Assembly: A versatile Golden-Gate framework towards universal DNA assembly, PLOS One