Apr 16, 2013

A Potential Standard for Constructing Modular Multi-Gene Expression Constructs

ACDC-SD and ToggleTec vectors: a new system for constructing modular multi-gene expression constructs

Organismal genes that encode integrated protein machines are frequently lumped into functional clusters, e.g. those involved in nonribosomal peptide synthesis (NRPS). The component proteins act in highly and tightly organized complexes to exert their physiological function.

The artificial integration of genetic function - such as production-optimized educt–product chains (e.g. in metabolic engineering) – at the functional level of biocatalysis is a challenge for synthetic biologists. The same holds true for probing and modifying structure-function relationships, e.g. when investigating intracellular or membrane-bound protein complexes that make up certain regulatory pathways.

This newsletter describes how the assembly / disassembly cloning and substitution design (ACDC-SD) principle and a series of corresponding kits for different expression hosts will help you tackle such challenges in building and efficiently modifying such multi-gene expression assemblies effectively.

ACDC-SD is fully compatible with and implemented in the Toggle assembly system (ToggleTec vectors). Any gene to be inserted into the vector thus needs to adhere to a limited number of design principles to exploit the full potential of the system (fig. 2).

Figure 1: [Click on image to expand] . Basic layout of Toggle vectors: A-boxes host applications which will generally be gene cassettes or gene clusters, S-boxes are selectors, T-boxes are target functions, e.g. transposition sequences, and M-boxes host maintenance functions, i.e. origins of replication for the host organism, etc.

ATG offers ready-made systems for E.coli, insect and mammalian cells but can also assemble custom-made expression systems tailored to your host of interest.

Advantages of ATG's fully design-optimized ACDC-SD gene cassettes:

  • Proven functional designs of gene clusters based on many years of expertise in constructional synthetic biology
  • Unified Design Concept to realize genes, expression boxes/ cassettes, regulators etc.
  • Designs incorporate a de-assembly option to regenerate individual expression boxes ? enables gene cluster revision and reorganization by assembling and/or reusing existing genetic elements (fig. between sites X and Y )
  • Option for substitution cloning: substitution of individual genetic elements or whole expression boxes in existing gene clusters (see fig. above)
  • Option for positional gene libraries for the local evaluation of variants in a given gene-cluster construct

Exchanging parts and modules in ACDC-SD designed gene cassettes Figure 2:[Click on image to expand] General lay-out of gene cassettes in the ACDC-SD. Functional and/or constructive elements such as regulators, promoters, leaders, coding sequence, terminators, etc. can be exchanged individually (colored double arrows and brackets at the top) or en bloc (dark blue double arrow and bracket at the bottom) by using design-inherent or specifically designed restriction sites.

ACDC-S design allows you to use standardized expression boxes where all genetic elements can be recycled and exchanged between synthetic biologists who use the same standards for different organisms. Swapping ACDC-S elements between gene clusters and researchers who work on the same project is easy.

All systems support a diversity of assembly strategies for the development of higher-order integrated genetic functionality, e.g.
  • Ligation strategies such as DONOR/ ACCEPTOR-based methods with alternating selector toggle assembly
  • Exonuclease cloning (5’ ? 3’ , 3’ ? 5’)
  • Recombination-mediated assembly, e.g. Cre/LoxP
Classical multiple cloning sites can be introduced into the systems as well.

ACDS-S is suited for these applications:
  • multi-gene artificial biochemical pathway systems – in vivo e.g.
    • anabolic pathways – biologically active compounds, fine chemicals, building blocks, biofuels etc.
    • catabolic pathways – biorefineries, bioremediation etc.
  • multi-gene hetero-protein-complex systemsin vivo
  • multiple in vitro bio-catalyst co-expression systems (flexiBAC)
  • other: specific functionally targeted multi-genetic constructive designs – in vivo
  • multiple-resistance mechanisms in plants etc.

Order your ACDC-S design for preassembled gene clusters of your choice or the special kits that allow you to assemble your gene clusters according to the ACDC-S design.

ATG is your “Synthetic Biology” system provider that provides fully integrated molecular service packages to help you improve the quality of your research and to accelerate your developments.

Shape the future world of molecular design … with ATG:biosynthetics!

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May 7, 2013
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PepID bio-based Peptide Libraries:



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