Overview

Anti-sense RNA is studied actively over the world. However, reliable method for gene silencing has not been clear. It is hard to find efficient sequence of anti-sense RNA and to synthesize new anti-sense fragment in keeping with your target gene.

fig1. you have to change anti-sense RNA conforming with each target gene.

We have a good idea! It is useful to use common anti-sense for different target gene.

fig2. Image of common anti-sense RNA effects.

Here we found that anti-sense-RBS (B0034) fragment, which is used by many iGEMers commonly, works to silence several proteins. We synthesized anti-sense B0034 fragment. Regardless of target gene, only one anti-sense fragment, anti-sense B0034, works on B0034 and repress the expressions of various proteins if they are regulated by B0034.

We also synthesized anti-sense-B0032 fragment in order to achieve specific gene silencing. You can change the target protein by changing the combination of anti-sense fragment and RBS locating upstream of target gene.

Specific anti-sense-RBS fragment helps you save labor to make new anti-sense RNA for each target genes. Fortunately, iGEM HokkaidoU team select tractable RBS for designing anti-sense-RBS fragment. You can use our anti-sense fragments without resynthesizing your constructs. What you only have to do is to add our anti-sense fragment to the construct with the target gene!!

fig3. Anti-sense B0034 has specific effects to B0034, RBS.

How to synthesize anti-sense constructs

Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-senes RBS was flanked with scar sequences. Moreover, the ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. After finishing synthesizing anti-sense RNA, we ligated anti-sense RNA with H-stem construction by NcoI and XhoI.

Fig. 1 How to make anti-sense B0034 by primer annealing

Fig. 2 Using restriction enzyme, XhoI, NcoI, we made stem_anti-sense conplex.

Fig. 3 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site

Fig. 4 B0034 & B0032 sequence

Fig. 5 Our parts

How to assay

We selected mRFP for target gene. We used fluorophotometer to measure how anti-sense worked. The colonies transformed by anti-sense RNA and target gene was used for assay.

1. To cultivate the colony in 4 mL LB culture for about 20 hours
2. To control turbidity up to 0.1 at OD600
3. To cultivate the colony in 2 mL M9ZB culture for 9 hours (IPTG induces antisense RNA, addition 20 uL)
4. To measure fluorescence after 9 hour

Fig. 6 Anti-sense B0034 is induced by IPTG