Part:BBa_K1323005

sRNA (1): YFP-targeting molecule

Small RNAs (sRNAs) are small double-stranded transcripts capable of silencing mRNAs of complementary sequence (Yoo et al., 2013). The sRNA sequence was designed with two functional parts: the part that is complementary to the target mRNA (target binding site) and MicC scaffold region for recruitment of Hfq (BBa_K1323019), a protein which enhances the sRNA to target mRNA hybridization (Yoo et al., 2013). The MicC RNA sequence is the best scaffold sequence as shown by previous studies done by the same group (Na et al., 2013). Since the target mRNA is YFP, the target binding sequence of this sRNA is complementary to the S. epidermidis codon optimized YFP (BBa_K1323010).

sRNA 1 target YFP gene starting at the end of its Shine-Dalgarno sequence. The SD region is determined by Yoo et al. to be the most effective binding region for mRNA silencing (Yoo et al., 2013). This 26 bp sequence has binding energy of -27.6 kcal/mol as determined using DINAMelt software (http://mfold.rna.albany.edu/?q=DINAMelt/Two-state-melting). For effective binding, sRNA should have binding energy between -30kcal/mol to -40kcal/mol (Yoo et al., 2013). We aligned sRNA1 sequence with other constructs and ATCC 12228 genome to determine the highest binding energy for non-specific RNA binding, which is -21.18 kcal/mol. This shows that sRNA1 binds more specifically to YFP and should not interfere with other gene expressions in our system.

This part was DNA-Synthesized.

References

Dokyun Na, S. M. (2013). Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nature Biotechnology, 170-174.

Na, D. et al (2013). Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nature Biotechnology 31, 170–174.

Yoo, S, M., Na, D., Lee, S. Y. (2013). Design and use of synthetic regulatory small RNAs to control gene expression in Escherichia coli. Nature protocol 8(9), 1694-1707.

Sequence and Features

Assembly Compatibility:

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

  Illegal XhoI site found at 140
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]