sRNA (2): 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: a region complementary to the target mRNA (target binding site) and MicC scaffold region for recruitment of Hfq BBa_K1323004, a protein which enhances the hybridization between the sRNA and target mRNA (Yoo et al., 2013). The MicC RNA sequence is the best scaffold sequence as shown by previous studies done by the same research group (Na et al., 2013). The target binding sequence of this sRNA is complementary to the S.epidermidis codon optimized YFP BBa_K1323010.
This part was DNA-synthesized by Bio Basic.
The targeting region of YFP.
sRNA 2 targets 5’UTR directly downstream of Shine Dalgarno sequence of the YFP construct. This 28 bp sequence has binding energy of -31.6 kcal/mol as determined using DINAMelt software (http://mfold.rna.albany.edu/?q=DINAMelt/Two-state-melting). For effective binding, sRNA should have a binding energy between -30 kcal/mol to -40 kcal/mol (Yoo et al., 2013). We aligned the sRNA 2 sequence with other constructs and the ATCC 12228 genome to determine the highest binding energy for non-specific RNA binding, which is -16.78 kcal/mol. This shows that sRNA 2 binds more specifically to YFP and should not interfere with other gene expressions in our system.
YFP cassette (pSB3K3 backbone, sarA protomer BBa_K1323021,TIR RBS BBa_K1323016, YFP cds BBa_K1323010)
sRNA 3 (BBa_K1323007)
Control sRNA (BBa_K1323008)
sRNAi Silencing assay
The YFP cassette and sRNA constructs were co-transformed into DH5α. The control for YFP expression included a sample with only the YFP cassette (no sRNA constructs), and a control sRNA that does not specifically target YFP. Colonies were streak purified and grown in LB supplemented with ampicillin. A restriction digest was run to confirm that both plasmids were in the colonies selected for the antibiotic resistance tests. Overnight cultures, in biological triplicate for each sample, were prepared. 50 µL of overnight culture was used to inoculate fresh media, and once the OD600 reached 0.5-0.6, the YFP fluorescence was measured.
Sequence and Features
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- 21INCOMPATIBLE WITH RFCIllegal XhoI site found at 142
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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.