Part:BBa_K5401006

Antisense Oligonucleotide System (ASO)

This composite part entails an antisense oligonucleotide (ASO) targeting the T7RNAP mRNA, coupled with a Hfq scaffold sequence, under the lac promoter. The various elements work hand-in-hand to regulate the expression of T7RNAP, thereby allowing the stable expression of T7RNAP in the bacterial host cell.

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. [BBa_K5401005]

Hfq is an RNA-binding protein that functions primarily as a riboregulator, facilitating the interaction between sRNAs and their target mRNAs. One of the key functions of the Hfq protein is RNA-RNA Interaction, through recognising its cognate Hfq scaffold sequence. [BBa_K5401004]

Usage and Biology

One of the key functions of the Hfq protein is to enhance the base-pairing between sRNAs and mRNAs, which can lead to the regulation of 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 our antisense oligonucleotide (ASO), 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
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 141
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]