Part:BBa_K3806018

RT primer/splint for W prefix addition (DRIVER)

The TU Delft iGEM 2021 project AptaVita looks at developing a modular, quantitative, and accessible rapid diagnostic test for vitamin deficiencies. In this project, vitamin-specific aptazyme biosensors are engineered through a novel in vitro evolution method, De novo Rapid In vitro Evolution of RNA biosensors (DRIVER) [1]. Range BBa_K3806017 - BBa_K3806021 [1] is a set of primers designed to assist the DRIVER selection process.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 32
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

DRIVER

Starting with a high-diversity library of potential biosensors, DRIVER makes use of a regeneration strategy within a directed-evolution procedure. This grants the selection of sequences that present a higher level of cleavage in the absence of ligand and a lower level of cleavage in the presence of ligand. During rounds in which the ligand(s) are present (also referred to as uncleavage selection rounds), the uncleaved RNA molecules preserve an unmodified 5’ prefix sequence from the previous round. These sequences are PCR amplified using this prefix and a specific suffix as primers. During rounds in which the target ligand(s) are absent (cleavage rounds), RNA molecules that undergo self-cleavage are regenerated by addition of a different 5’ prefix. Afterwards, these sequences are PCR amplified using the newly added prefix and the specific suffix as primers. By iteratively applying this process, each round selectively enriches the desired subset of sequences from the library. Eventually, RNA sequences whose cleavage is regulated by the target ligand(s) dominate the population.

BBa_K3806018 in DRIVER

Following the cleavage step where the DNA library is transcribed to RNA and the aptazymes are allowed to self-cleave, BBa_K3806018 is used in a RT step to create a complementary DNA (cDNA) product. The same oligonucleotide guides the repair of the cleaved aptazymes during a ligation step, resulting in a sequence with a different 5′ prefix, called W prefix.

Sequence: /5’PHOS/GACAGCTTTGTTTGTTTCCCAAGCUGTCACCGGATCCGGTCTGATGAGUCCTTTCTTTTTGCTGTTTCGTCC 3’

It is important to note that the primer should be 5′-phosphorylated for ligation to occur. Additionally, this primer contains two uracil bases. A Uracil-Specific Excision Reagent (USER) is used to cut the cDNA at these two locations, resulting in a linear DNA product with the newly incorporated W prefix.

References

1. [1]Townshend, B., Xiang, J. S., Manzanarez, G., Hayden, E. J. and Smolke, C. (2021). A multiplexed, automated evolution pipeline enables scalable discovery and characterization of biosensors. Nat Commun, 12, 1437.