A Flexible System for Applications in Synthetic Biology
Synthetic biology combines molecular biology and systems biology with engineering principles to design and create biological systems not found in nature. In biotechnology, the design of dedicated bio-factories with improved or tailor-made biological functions to fight current and future challenges is one aim of development. Creation of functional, self-regulating microorganisms for use in bio-transformations of renewable resources into economically attractive compounds requires a careful combination of biochemical characteristics from various donor organisms with genetic regulatory systems of a dedicated host.
Regardless of size or destination, synthetic biology starts with comparably small information units, which need to be combined and properly arranged in order to achieve a certain goal. This may be the de novo synthesis of individual genes from oligonucleotides, a shuffling of protein domains in order to create novel biocatalysts, the assembly of multiple enzyme encoding genes in metabolic pathway design or strain development at the production stage. Sooner or later, individual genetic information units are combined with promotors, terminators and other regulatory elements in order to permit expression in heterologous systems. Thus, a useful cloning system for application in synthetic biology must provide easy-in and easy-out capabilities.
The CoLibry toolbox consists of zero background vectors and is available with ampicillin, chloramphenicol, kanamycin or streptomycin resistances for selection. At present, a total of 16 vector backbones are available, which combine 4 origins of replication (RSF, ColE1, CDF, p15A) with the resistance genes. Tailor-made derivatives of core vector backbones are readily accessible on demand, which provide various endonuclease cleavage sites for accommodation or release of foreign DNA.
The primary cloning vectors are designed for the accommodation of blunt-ended DNAs. The inserts are either provided by PCR or by pre-annealed synthetic oligonucleotides. The vectors provide class IIS endonuclease (CTSE) recognition sites located in order to release the inserts in subsequent steps (fig.
1A), whereby the terminal base pairs form unique combinatorial sites: Depending on the CTSE used, 3 (LguI) or 4 (BpiI, Eco31I, Esp3I) bases long 5’-overhangs are generated upon cleavage. Whenever the generated overhang at the 3’-end of the upstream fragments fits the overhang of the 5’-end of the downstream located one, these fragments are readily fused in an ordered and oriented manner.
Secondary cloning vectors are used for the accommodation of multiple inserts from primary libraries or previous level fusion vectors. These vectors provide two pairs of CTSE recognition sites for level fusion cloning. The accommodation sites are usually generated by the same CTSE which is used to release fragments from donor vectors in a given experiment and provides compatible ends for the 5’-overhang of the first fragment and the 3’-end of the last fragment assembled. Number and sequence of internal fusion sites are freely selectable as long as unique overhangs are generated at each fusion site. Thus, the utilization of a single CTSE in a cloning step and the lack of its recognition sites in the resulting vector permit a single cup reaction for cleavage and ligation of the resulting fragments, which proceed simultaneously in an ordered an directed manner (fig. 1B). A second pair of convergently oriented CTSE recognition sites permits the release of the fused DNA fragment in subsequent steps. Applying multiple rounds of fusion, the system allows merging of genetic information to the practical reception limits of the plasmids up to 25-30 kb.
The toolbox can effectively aid cloning of large protein encoding genes or other genetic elements. Initially amplified and cloned in smaller fragments of usually up to 1.5 kb, the final sizes of the inserts can exceed 10 kb. It is worth noting that these large fragments are possibly released by CTSEs with 4 bases long 5’-overhangs compatible for accommodation by class IIP endonucleases frequently found in the multiple cloning sites of plasmids, cosmids, phages or BACs. This permits assembly and release of DNAs containing e.g. multiple NcoI and XhoI sites within the sequence using Eco31I, Esp3I or BpiI to provide the compatible ends for the accommodation by NcoI/XhoI cloning sites in the accommodating vector (fig. 2A).
Likewise, protein encoding genes of all sizes can be fused in combinatorial approaches with various partners providing affinity tags for facile purification, solubility improving fusion partners, fluorescent proteins for in vivo tracing of the target protein or other genetic elements including regulatory sequences or ribosomal binding sites. Furthermore, smaller fragments are readily provided by pre-annealed oligonucleotides, which provide the required overhangs (fig. 2B). This flexibility permits a systematic and cost effective approach for an ordered identification of optimum solutions for given problems. Obviously, individual genes can be combined with different promotors in order to allow an ordered expression of multiple genes from a single plasmid. Last but not least, up to four individually generated compatible plasmids can be generated and combined in a single cell in order to study complex interactions.
The applications of the toolbox are not just limited to molecular biological applications but also allow and facilitate a variety of applications in synthetic biology, which have previously only been achieved with considerable effort and time, or shaped up as entirely unrealizable. The strength of the method lies in the possibility to use individual DNA fragments, which are initially cloned and sequenced in order to be used in multiple subsequent applications. An obvious example for this feature is the initial cloning of enzyme encoding genes, which are subsequently used in order to generate multiple variants which facilitate purification and detection, or to improve solubility in multiple downstream applications.
This can either be done by genetic in frame fusion of the target sequence with well characterized fusion partners, or in random approaches, where hitherto not characterized elements are combined with the target in combinatorial approaches. An example for the latter is a screen of host or tissue specific promotors, which are readily placed in compatible libraries, too. Given that such optimum solutions can be individually found for multiple genes, which are then combined in larger assemblies or combined from individual vectors in vivo or in vitro, the value of the method is further increased.
Two recently published studies (Schiffels, Pinkenburg et al. 2013, Schiffels and Selmer 2015) have clearly shown that the combination of the novel cloning strategy for generation of multiple enzyme variants is very useful in order to provide the building blocks for combinatorial analysis: The synthesis of combinatorial plasmid constructs did not only enable the rapid generation of co-expression plasmids for a total number of 14 genes, but also offered rapid access to plasmids lacking individual constituents, which were then supplied independently in plasmids providing the required genes for full reconstitution. Notably, the reconstitution was achieved in vivo when libraries of putative lacking elements were introduced into the same cell by a second plasmid, but also when prepared cell extracts containing individually produced elements of the system were combined in order to restore the natural system in vitro.
The latter approach was particular useful in order to establish metabolic bottlenecks and other limitations in a [NiFe]-hydrogenase maturation system and facilitated process optimization, while the former approach enabled large scale production of functionally matured hydrogenase in a heterologous system. The possibility to create affinity tagged variants of the target enzyme or individual constituents involved in maturation did not only allow rapid purification of the target enzyme in good yields but also effectively enabled the design of pulldown experiments to study specific interactions between the target enzyme and its maturation factors.
Whereas the system in its present form is an effective tool to study complex enzyme maturation or metabolic pathway design in a laboratory setup and uses modular genetic units which are readily combined on demand, applications in synthetic biology generally require genomic manipulation of dedicated hosts once the process reaches production stages. The introduction of multiple large DNA assemblies into the genome of organisms is still a tedious, time-consuming and often futile effort. Our current efforts are therefore focused on the development of a CoLibry-based system for rapid and sequential integration of multiple modules into the genome of the host. In a sequential approach, a stepwise introduction of individually generated and tested metabolic modules will allow stepwise generation of production strains according to the needs of various interests, e.g. in the development of solvent producing organisms.
 J. Schiffels et al.: PLoS One 8 (7): e68812 (2013)
 J. Schiffels and T. Selmer: Biotechnol. Bioeng. (2015) DOI: 10.1002/bit.25658 [Epub ehead of print]
Prof. Thorsten Selmer
Institute of Nano- and Biotechnologies
Aachen University of Applied Sciences
Department of Chemistry and Biotechnology
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