Part:BBa_K1017202

Regulation RBS-2

Description of function

sRNA are small (50-250 nucleotide) non-coding RNA molecules produced by bacteria; they are highly structured and contain several stem-loops.sRNAs interact with the targeted mRNAs by imperfect base pairing, occluding the Shine-Dalgarno sequence thus prevent the ribosome from binding to the initiation condon, so the translation would be repressed.

In our project we have designed the artificial sRNA(BBa_K1017404[1]), which contains a consensus sequence, 5’-CCCUC-3’, that can base pair with the SD sequence due to complementarity, and we can get a new RBS which only would be bound with our artificial sRNA. Here we call this RBS as rRBS.

We designed the rRBS by employing the sRNA targeting region from ompF so that the sRNA would only complementary with ompF but not the others in the E.coli. We also make the AUG codon sufficiently apart from the SD sequence for ribosome binding. Therefore, other iGEM teams can use this sRNA regulation system in there project by adding this RBS to the upstream of any gene they want to regulate.

The following figure depicts the sRNA specifically binds on rRBS by base pairing, forming a blockade of ribosome binding. Therefore, the translation is inhibited.

Quantitative data showing the Part or Device function

We used the following biobrick to test the translational efficiency of K1017202 (rRBS) compared to the efficiency of other RBSs:

   Pcons + BBa_B0034 + mRFP + Ter
   Pcons + BBa_K1017202+mRFP+Ter
   Pcons + BBa_B0030 + mRFP + Ter
   Pcons + BBa_B0032 + mRFP + Ter
   control: pet 30 

As you can see from the figures, the bacterial pellet and liquid of each biobrick shows different level of RFP expressions as the RBS of each biobrick provides a different translation efficiency. The deeper the red color is, the higher the level of expression is.

Acknowedgment of sources and references

Torsten Waldminghaus, Nadja Heidrich, Sabine Brantl and Franz Narberhaus .(2012). Engineering Artificial Small RNAs for Conditional Gene Silencing in Escherichia coli ,1: 6–13

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]