Team:BioBricks/Design
The wetlab part of our project focused on designing a polycistronic design with precise expression levels and low complexity of regulation unit to enable forward engineering of modules. Here we got inspired by natural operons realizing transcriptional and translational coupling by frameshifts of stop-start-codon and integrating RBSs in upstream genes. Based on prior research by Mutalik et al (2013) on bicistronic design we searched for an convincing process to integrate RBSs in upstream genes to realize polycistronic designs with frameshifts and no interregional peptides. We succeeded in designing this construct by using a leader peptide to enable translational coupling for the first gene and then integrating all following RBSs into the corresponding upstream genes. This construct is called the “Daisy Chain”.
How to Construct a Daisy Chain
In order to realize a Daisy Chain one has to optimize the order of genes in the operon based on possible Shine-Dalgarno Sites and search for codon changes to embed SDs and thus increase/ decrease translation. To identify potential embedded Shine-Dalgarno sites and measure the strength based on sequence, codon-changed sequence, and position of SD as well as upstream and downstream regions, we are using an Excel tool coupled with the database and calculation algorith of EMOPEC. To increase the range of expression levels for a given operon, both SD sequence can be varied in strength and the N-terminal end of the gene can be codon optimized to increase translation rate (Goodman 2013) within the Excel tool.
Advantages
- Combines transcriptional regulation of multiple genes for an easy configuration
- Achieves similar transcription levels within the operon while having a compact architecture with less parts and less potential repetitive regions.
- Translational coupling unfolds secondary structure to enable reliable translation of the downstream gene.
Disadvantages
- Transcriptional efficiency may vary with the length of the sequence
- Reduced range of expression for linked genes
- Coupled genes are regulated together within a single operon
- The ability to embed an SD in the upstream gene is dependent on codon degeneracy
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