Part:BBa_K5401005

T7 RNA polymerase (T7RNAP) antisense oligonucleotide (ASO)

The antisense oligonucleotide is a short RNA (sRNA) sequence complementary to the T7RNAP mRNA sequence. It binds to +4 to +27 of the mRNA sequence, thereby inhibiting translation and regulating the translation of T7RNAP.

The DNA sequence provided transcripts an sRNA sequence complementary to the wild-type T7RNAP (BBa_J435007).

Usage and Biology

The aim of the antisense oligonucleotide is to bind to the T7RNAP mRNA and inhibit the translation.

In our design: the Hfq protein binds to sRNAs (an antisense oligonucleotide targeted against T7RNAP mRNA), thereby helping them to recognize and bind to complementary regions on mRNAs. This binding blocks ribosome access to the mRNA's start codon or Shine-Dalgarno sequence, thus inhibiting translation. This helps to knockdown the expression of T7RNAP, thereby regulating the expression of T7RNAP.

Characterization

This part, together with a Hfq scaffold sequence, and a lac promoter acts to regulate the expression of T7RNAP, thereby allowing a stable expression. When the ASO system was cloned into the T7-expressing plasmid (plasmid 1c), the expression was T7RNAP was observed to be more stable, with lower frequency of insertions as compared to the predecessor plasmid (plasmid 1a).

Fig 1: Colony PCR results showed the presence of insertion sequences within the coding sequence of T7RNAP for E. coli reporter cells transformed with plasmid 1a. Band size corresponding to ~3000bp (different from expected band size of 1892bp) was observed for all 8 colonies picked from reporter cells transformed with plasmid 1a, thereby showing the unstable expression of T7RNAP. Band size corresponding to ~2000bp (expected band size of 2038) was observed for 7/8 colonies. for E. coli reporter cells transformed with plasmid 1c. The lower frequency of insertion observed shows a more stable expression of T7RNAP.

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]