Combinatorial parts assembly (CPA) is a way to combine predefined DNA parts (e.g., promoters, ribosomal binding sites, open reading frames, terminators, etc.) in order to build a diverse set of larger constructs. Users simply provide individual part sequences and progression of the parts within the order form. The final construct is synthesized, with sequence junctions and frame all handled seamlessly by the assembly.
All conceivable part combinations can be created to build and test new metabolic pathways or a variety of expression cassettes to identify the most valuable combination.
Deliverables and storage
Combinatorial part assemblies are available as separate constructs or pooled libraries. Depending on the diversity (number of theoretical combinations possible) of your library we offer two options:
- 5 µg of plasmid DNA will be delivered per construct
Application example (CPA)
Optimization of heterologous protein expression is not just a matter of gene sequence optimization for the host organism. Tuning the expression level by choosing the optimal promoter and terminator combination is also an essential part of an expression project. For example, high levels of foreign proteins caused by a strong promoter or insufficient termination by a weak terminator can lead to growth inhibition of the host.
To demonstrate this concept we generated a combinatorial library composed of a small set of yeast promoters and corresponding terminators from the GeneArt® Elements™ part collection to analyze relative luciferase expression levels (Figure 1). In this experiment, the highest firefly luciferase expression was shown using a TEF1 promoter/TEF1 terminator combination (Figure 2). This demonstrates the proof of concept, and can be extrapolated to test other functional elements, such as in metabolic pathways.
|Figure 1. Yeast constitutive promoters and terminators from the GeneArt® Elements™ CPA collection used for creation of the combinatorial promoter–terminator library.|
Figure 2. Twenty-five different promoter/terminator combinations were created to control firefly luciferase reporter expression. Reporter expression levels were measured and normilized against co-transformaed renilla luciferase driven by a standard promoter.
Frequently asked questions (FAQs)
Q. Can CPAs also be ordered via the GeneArt® portal?
A. No. Currently orders can only be fulfilled by completing the questionnaire and sending it firstname.lastname@example.org
Q. How many different combinations of parts can you provide?
A. Pooled libraries provide up to 10e6 different constructs.
Q. What is the delivery time for a CPA project?
A. The delivery time is very project specific. It largely depends on the complexity of the project (how many variants are being requested, how many building blocks need to be created, etc.)
Q. How do you quality control the libraries?
A. That depends on the nature of the deliverables. If you request separate constructs, those will be completely sequence verified. However, if the diversity of the library is very high (e.g., >10e4) and a pool of clones will be delivered, and a peer-group of library specimens will be sequenced in order to verify the library quality.
Q. Do you provide CPAs in formats other than E. coli plasmids or cells?
A. No, we are currently using only E. coli in our standard production process. That means that the plasmids we are using need to be at least shuttle vectors that allow for the propagation in E. coli.