Part:BBa_K3192000

Custom RBS Optimized for BBa_K3192020

The 2019 Virginia iGEM Team sought efficiency and productivity in designing their genetic construct as the device required two plasmids to operate. Two plasmids bears a large metabolic load on the chassis¹, so, synthetic ribosomal binding sites were developed in order to optimize translations rates for the genes used in the styrene-degrading and polyhydroxybutyrate-producing plasmids. This ribosomal binding site is optimized for part BBa_K3192020 with a translation initiation rate of 666901.45.

When expressing gene clusters in a foreign chassis, the endogenous ribosomal binding sites (RBS) are often included in the sequence information provided by NCBI. Although these RBS are sufficient, they may not provide optimal translational efficiency. Synthetic RBSs provide a solution to increase translation initiation rates by using software to design a RBS with a linear secondary structure that allows for optimal ribosomal binding and translation. Our team’s project required enhanced translation of our coding sequences; therefore we chose to optimize all RBSs for high translation initiation rates. Using DeNovo DNA, our team created synthetic RBSs that ran thousands of iterations to create substantially elevated translation initiation rates.

Design

Denovo DNA was used for design of the Ribosomal Binding site. Given the coding sequence for the specified gene, DeNovo ran thousands of iterations to develop a RBS with high translation initiation rates. This sequence was then synthesized (for the 2019 Virginia iGEM team it was synthesized with its respective coding region). Sequencing confirmed the identity of the synthesized construct.

References:

1) Watve, M. M., Dahanukar, N. & Watve, M. G. Sociobiological control of plasmid copy number in bacteria. PloS one (2010). Available at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2827543/. (Accessed: 22nd October 2019)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]