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 that matches your target gene.

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

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. All you have to do is to add our anti-sense fragment to the construct with the target gene!!

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.

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, add 20 uL)
4. To measure fluorescence after 9 hour

Preliminary results

We experimented whether B0034antisense worked specifically by expressed antisense RNA and used Nakashima's plasmid(pHN1257) as a vector. We double transformed separate plasmids of antisense and target gene and assayed them. All antisenses are on pHN1257 and all target genes are on pSB6A1.

Our samples are following four.

target B0034+ antisense B0034

target B0034+ antisense B0032

target B0032+ antisense B0034

target B0032+ antisense B0032

We examined each samples with And without IPTG induction.

The way of assay is following.

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 2 mL LB culture for about 18 hours
2. To control turbidity up to 0.1 at OD600
3. To cultivate the colony in 2 mL M9ZB culture for 18 hours (IPTG induces antisense RNA, add 20 uL)
4. To measure fluorescence after 18 hours

Conclusion

We were able to confine the specific work of antisense in case using Nakashima’s stem. On the other hand, in case of H-stem, we could confirm only transcription of antisense but we could not get a proof that antisense worked specifically by our experiments. We want to show some results by presentation in Boston by rethinking copy number of plasmids or medium for assay to work antisense even in H-stem.

Instablity of mRFP expression

It’s possible to suppress 80% of registered parts in iGEM using RBS by asB0034 and asB0032. In short, it is very provable to be a common way of expression of proteins because it can suppress iGEM parts easily.