APPLICATIONS OF TECHNOLOGY:
- Biotechnology research and development
- Biofuels research and development
- Reduces the labor associated with current technologies
- Determines cost-minimizing DNA assembly strategies
- Condenses multiple projects for use with high-throughput platforms
Researchers at the Joint BioEnergy Institute (JBEI) have developed j5, a technology that automates and optimizes the design of the molecular biological process of cloning/constructing DNA. j5 enables users to benefit from multi-part scar-less SLIC, Gibson, CPEC, (combinatorial) Golden-gate assembly, or variants thereof, where automation software does not exist, without the current level of intense labor associated with the process. The j5 manual provides a step-by-step user’s guide and describes the software’s feature set in greater detail.
The JBEI technology inputs a list of the DNA sequences to be assembled, along with a genbank or FASTA format sequence file for each DNA source. A BioCAD software package, a canvas for arranging iconic representations of biological parts that allows the user to select, order, and manipulate the parts with drag-and-drop simplicity, provides a graphical user interface that facilitates and automates the preparation of the j5 input files. Given the list of DNA sequences to be assembled, j5 first determines the cost-minimizing assembly strategy for each part (direct synthesis, PCR/SOE, or oligo-embedding), designs DNA oligos with Primer3, adds flanking homology sequences (SLIC, Gibson, and CPEC; optimized with Primer3 for CPEC) or optimized overhang sequences (Golden-gate) to the oligos and direct synthesis pieces, and utilizes BLAST to check against oligo mis-priming and assembly piece incompatibility events.
After identifying DNA oligos that are already contained within a local collection for reuse, the program estimates the total cost of direct synthesis and new oligos to be ordered. In the instance that j5 identifies putative assembly piece incompatibilities (multiple pieces with high flanking sequence homology), the program suggests hierarchical subassemblies where possible. The program outputs a comma-separated value (csv) file, viewable via Excel or other spreadsheet software, that contains assembly design information (such as the PCR/SOE reactions to perform, their anticipated sizes and sequences, etc.) as well as a properly annotated genbank file containing the sequence resulting from the assembly, and appends the local oligo library with the oligos to be ordered. Although not currently implemented, j5 will soon output the genbank, assembly csv file, and other requisite information directly to a parts registry, e.g. the JBEI Registry, which further reduces the labor of the process, as well as condense multiple independent assembly projects into 96-well format for high-throughput liquid-handling robotics platforms.
DNA assembly, or cloning, has traditionally been accomplished by inserting several DNA sequences via restriction digest and ligation into the multiple cloning sites of a plasmid vector. As the size and number of the inserts to be incorporated into the plasmid increases, so does the difficulty of construction. The process must be repeated from the starting point for alternate combinations of inserts. Standardized DNA assembly protocols, such as the BioBricks method, have recently been developed to facilitate the process and promote the reuse of previous construction efforts.
However, some standardized methods including BioBricks retain several drawbacks such as the introduction of undesirable scar sequences and technical details that limit the combinatorial complexities of derived plasmid libraries. Several alternative DNA assembly protocols, including multi-part SLIC, Gibson, CPEC and Golden-gate, do not have these disadvantages but require time-consuming design of DNA oligos that either contain flanking homology sequence to neighboring parts (SLIC, Gibson, and CPEC) or specified overhang sequences (Golden-gate), and also require validation processes that safeguard against oligo mis-priming and the assembly piece incompatibilities that often arise from the inclusion of homologous sequences. j5 automates the requisite oligo design and validation processes associated with the SLIC, Gibson, CPEC and Golden-gate DNA assembly methodologies.
The Joint BioEnergy Institute (JBEI, www.jbei.org) is a scientific partnership led by the Lawrence Berkeley National Laboratory and including the Sandia National Laboratories, the University of California campuses of Berkeley and Davis, the Carnegie Institution for Science and the Lawrence Livermore National Laboratory. JBEI’s primary scientific mission is to advance the development of the next generation of biofuels.
STATUS: Patent pending and copyrighted. The web-based version of j5 is available for non-commercial users under a no-cost license agreement. For more information, visit the j5 website. Commercial distribution rights have been licensed to TeselaGen Biotechnology, Inc.
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REFERENCE NUMBER: ECRB-2836, EIB-2928