Team:Duesseldorf/MoClo

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• Modular Cloning
• Cyano Q&A

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  • Plant Synthetic Biology
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  • Modular Cloning
  • Cyano Q&A
• Judging Form

Modular Cloning

The modular cloning techniques allow the assembly of different DNA fragments such as promoters, ribosomal binding sites or terminators. It is based on the ability of Type II S restriction enzymes to cleave outside their recognition sequence. That way sites with compatible overhangs can be assembled as desired. The advantage of this method is the cloning of entire transcriptional units in a single reaction. This is granted by the Golden Gate cloning system. The kits are arranged in a way that the scientist designs his part in different cloning vectors, from so called Levels 0 to 2. This includes the construction of single basic parts (Level 0) to a complete transcriptional unit (Level 1). In the end more complex modules can be built by the assembly of different transcriptional units (Level 2) and so the formation of elaborated genetic circuits is enabled¹.

Our iGEM team this year is making use of two different modular kits: Since cloning of Saccharomyces cerevisiae as one of the three organisms of the co-culture is necessary, the MoClo-YTK kit is used for cloning purposes². As previously described, this kit contains standardized part sequences, which can be assembled in vectors of different levels. The peculiarity of this tool collection is the possibility to easily clone in Escherichia coli, select and choose the correct clone in this bacterium, as the bacterial origin of replications can be added. Consequently, the appropriate construct can be transformed in yeast as well and selected based on the presence of an amino acid encoded in the construct ³.

In addition to that, E.coli represents one of the three organism in the co-culture that is tried to be established. Hence the availability of the CIDAR MoClo Parts kit is exploited ⁴. Similarly, this kit allows easy, fast and one pot reactions of different, complex constructs. Moreover, the switch of different antibiotic resistances allows the selection at different levels, reducing the amount of false positive clones throughout the different levels ⁵. Those systems are very simple to use, as they are supplied with sufficient supplementary material that explains the cloning procedures in detail, including suitable reaction conditions as well as protocols for the Golden Gate reactions. In case of high cloning amounts, these toolboxes can drastically facilitate the experiments and improve final results.

References

1. https://www.addgene.org/cloning/moclo/
2. https://www.addgene.org/kits/moclo-ytk/
3. Lee, Michael E., et al. "A highly characterized yeast toolkit for modular, multipart assembly." ACS synthetic biology 4.9 (2015): 975-986.
4. https://www.addgene.org/cloning/moclo/densmore/
5. Iverson, Sonya V., et al. "CIDAR MoClo: improved MoClo assembly standard and new E. coli part library enable rapid combinatorial design for synthetic and traditional biology." ACS synthetic biology 5.1 (2015): 99-103.