Overview

Anti-sense RNA is studied actively over the world. asRNA can be easily synthesized, but there is no clear method to make stable, highly efficient asRNA. It is a labor to design efficient asRNA for every target gene you want to repress.

Therefore, we decided to design an universal asRNA which could repress various target genes.

We found that anti-sense RBS (B0034) fragment, which is used by some 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

As shown in Fig. 2, we were able to see asB0034 and asB0032, induced by IPTG, working to the target, B0034. However, toward B0032, neither asB0034 nor B0032 was confirmed working. From these results, we were not able to confirm specificity of asB0034, but toward the construct B0034, asB0034 down regulated the expression 40%, and asB0032 showed the regulation of 80%. Nakashima gained a regulation of 78%, so we were able to get an equal result.

Result

We inserted antisense on H-stem and assayed them to make antisense working in case H-stem not Nakashima's stem. All antisenses are on pSB1A3 and all target genes are on pSB4C5. Samples are following

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 4 mL LB culture for about 22 hours
2. To control turbidity up to 0.1 at OD600
3. To cultivate the colony in 2 mL M9ZB culture for 30 hours (IPTG induces antisense RNA, add 20 uL)
4. To measure fluorescence after 30 hours

From this experiment, we were not able to confirm whether anti-sense is working by IPTG induction using fluorescence intensity. So, we checked the expression of anti-sense using RT-PCR, and one with IPTG induction showed the expression of anti-sense. (However, we were unable to gain a data of IPTG-.) From this result, we confirmed that, though not largely, asB0034 and asB0032 worked toward B0034.

Discussion

1. Anti-sense was not induced by IPTG; it is leaking.
Seen from Fig. 3, fluorescence strength did not differ between IPTG+ and IPTG-. Since fluorescence showed no difference, it could be assumed that anti-sense was expressing regardless of IPTG induction. Also, on Fig. 1, it was confirmed that asB0034 works, but it showed no activation on H-stem. Therefore, because anti-sense was not under regulation of IPTG induction, we were not able to confirm the activity of anti-sense by fluorescence intensity.
2. Antisense did not show any expression
Although it would be a contrasting discussion to discussion 1, from fig.1, we could not find little gap in fluorescence of mRFP in antisense B0032 with/without IPTG inducing. Likewise from fig.3, we found little gap either. In consideration of these facts, we guessed that antisense did not express. We confirmed the existence of antisense B0034 by sequencing, though we did not about antisense B0032. The reason is that it is so difficult to sequencing of DNA which had stem-loop stractures.
3. Instability of copy number of target gene
Seeing Fig.1and 3, even if it were the same target genes, they sometimes had big differences in the degree of expression. By all rights, target gene under the control of B0034 which is stronger RBS should be larger degree of expression than that of B0032. But the result was completely opposite. Owning to making several assays, target gene increased little or in case, even if the same origin of plasmids, cultivate them from different colony, the expression of mRFP showed gap. Therefore, because of changes of copy number of target gene, expression of target genes weren’t enough and we found that it was difficult to measure and estimate the activation of antisense by fluorescence.

Improvement

1. Analysis by RT-PCR
By analyzing quantity of anti-sense RNA, we realize whether anti-sense is induced by IPTG adding, without IPTG. We can realize anti-sense work even if copy number is unstable. We compare mRNA of target gene with mRNA of target gene with double transformed anti-sense.
2. Improvement of medium to induce anti-sense by adding IPTG easily
To induce anti-sense RNA by IPTG easily, we have used M9ZB culture. However the medium includes glucose, IPTG induction may be too late. We try to use LB medium to realize IPTG induction.
3. Stability of copy number in target gene
It was published that low copy plasmid often occur movement of copy number. We need to select higher copy number. We use medium included an 1.5 times antibiotic for screening.

We are going to show positive result in Boston!

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.

Instability 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.


Fig. 6 Rate of RBS used BioBrick parts