Designed by: Wen-Hui Cheng   Group: iGEM13_NCTU_Formosa   (2013-09-12)

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.

Figure 1. Hfq and a small RNA may sequester the ribosome-binding site of a target mRNA, thus blocking binding of the 30S and 50S ribosomal subunits and repressing translation.

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:

  1. Pcons + BBa_B0034 + mRFP + Ter
  2. Pcons + BBa_K1017202 + mRFP + Ter
  3. Pcons + BBa_B0030 + mRFP + Ter
  4. Pcons + BBa_B0032 + mRFP + Ter
  5. 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.

Figure 2. B0034 clearly expressed more RFP compared to others.
Figure 3.K1017202 shows moderate RFP expression that can be distinguied from the expressions B0030 and B0032.
Figure 4.From left to right, the ribosome biding sites respectively: B0032, K1017202, B0030, B0034, control.

We measured the normalized expression for each biobrick mentioned above. We calculated the normalized expression by dividing fluorescence expression with the OD value measured, since the higher the OD value is, the larger the amount of bacteria that can express florescence would be. As shown in the chart, the normalized expression of the biobrick with B0034 is the highest and the one with K1017202 (rRBS) is the second highest, while the other two of B0032 and B0030 show weak expressions. This result implies that K1017202 can, in fact, serve as a functional RBS. In comparison to other RBS, K1017202 can provide moderate translational efficiency that is just lower than that of the highly efficient B0034.

Figure 5.K1017202 shows moderate expression compared to the others.

Then, We employed the following biobricks to test the regulation efficiency of the sRNA we designed :

Figure 6.The biobrick of testing the sRNA.

From the following figure, it can be observed that sRNA can regulate RFP expression. The bacterial colonies without sRNA are red, showing high RFP expression. The bacterial colonies with sRNA, however, are white, suggesting that sRNA has regulated the RFP expression by base pairing to rRBS. The normalized expression without sRNA must be much higher than the expression with sRNA, or else we would not be able to easily distinguish the difference in RFP expression through the colors of the colonies.

Figure 7.The bacterial colonies with sRNA shows no clear sign of RFP expression, while the colonies without sRNA do.

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
  • 12
  • 21
  • 23
  • 25
  • 1000