Team:HokkaidoU Japan/Projects/asB0034

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Projects
Anti-Sense B0034

Overview

Anti-sense RNA (asRNA) 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.

Fig. 1 You have to modify asRNAs conforming with each target gene.

As a solution, we decided to design a general asRNA which could repress various target genes.

Fig. 2 Concept of common anti-sence sequence.

To achieve this, we constructed an asRNA for RBS. For gene expression, RBS is indispensable and most of iGEMers use B0034 in their projects. Thus, we constructed and registered an asRNA for B0034 as a new part "Anti-sense RBS fragment B0034 (BBa_K1524107)". By this part, iGEMers can repress any target gene that is synthesised downstream B0034.

We also registered an anti-sense RBS fragment for B0032 "Anti-sense RBS fragment B0032 (BBa_K1524108)". You can repress the target gene individually by changing the combination of anti-sense RBS fragment and the target gene.

You can repress expression of your target gene without resynthesizing your constructs. All you have to do is to add our asRNA to the construct with the target gene!!

Fig. 3 Anti-sense B0034 has specific effects to B0034, RBS

Method

This is how to synthesize anti-sense constructs. Their targets are B0034 (BBa_B0034) and B0032 (BBa_B0032). Both RBS are popular among iGEM. Anti-sense RBS fragment was synthesized by primer annealing. Based on BioBrick standard, anti-sense RBS was flanked with scar sequences. The ends of anti-sense fragment have restriction enzymes recognition sites, NcoI and XhoI. Therefore, after the synthesis of anti-sense RNA, we can ligated asRNA with H-stem construct by NcoI and XhoI.

Fig. 4 How to make anti-sense B0034 by primer annealing

Fig. 5 Using restriction enzyme, XhoI and NcoI, we made stem_anti-sense complex.
Fig. 6 Blue; antisense B0034, B0032 Red; scar sequence Green; NcoI site Purple; XhoI site
Fig. 7 B0034 & B0032 sequence
Fig. 8 Our parts

How to assay

We selected mRFP as the target gene. We used fluorophotometer to measure how the asRNA worked. The colonies transformed by asRNA and the target gene were used for the assay.

  1. Cultivated the colony in LB medium.
  2. Centrifuged the culture at 10,000 rpm / for 2 min / at 25°C
  3. Removed the supernatant and add M9ZB medium then voltex the pelet.
  4. Performed RT-PCR
  5. Measured absorbance of 260 nm about cDNA.
Fig. 9 Anti-sense B0034 is induced by IPTG

Preliminary results

We assessed whether anti-sense B0034 works as asRNA by using Nakashima’s plasmid (pHN1257[1][2]) as a vector. We double transformed separate plasmids of antisense and target gene and had an assay. All anti-sense constructs are on pHN1257 and all target constructs are on pSB6A1.

Details of pHN1257 vector

This vector is published by Nakashima for transcribed anti-sense RNA. The feature of vector is Paired Termini (PT) design. PT makes stem-loop construct and stabilizes anti-sense cassettes. The restriction enzyme (NcoI and XhoI) sites are between PT. The vector resistance is Kanamycin, has IPTG induce promoter, Ptrc and 30 copy numbers (reprication origin is pSC101).

Our samples are following

  • target B0034+ anti-sense B0034
  • target B0034+ anti-sense B0032
  • target B0032+ anti-sense B0034
  • target B0032+ anti-sense B0032

We examined each samples with / without IPTG induction.

The way of assay is following.

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

  1. Cultivated the colony in 2 mL LB culture for about 18 hours
  2. Controled turbidity up to 0.1 at OD600
  3. Cultivated the colony in 2 mL M9ZB culture for 18 hours (IPTG induces antisense RNA, add 20 uL)
  4. Measured fluorescence after 18 hours
Fig. 10 Fluorescence strength of each sample with / without IPTG
Fig. 11 Rate of IPTG(+) and IPTG(-). Numeric became small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression.

As shown in Fig. 11, we were able to see the translation of 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 were down regulated the expression 40%, and asB0032 showed the regulation of 80%. Doc. Nakashima gained a regulation of 78%, so we were able to get the same result.

Result

We inserted anti-sense into H-stem and assayed them to make anti-sense working in not only Nakashima’s stem but on our own H-stem. All anti-senses are on pSB1A3 and all target genes are on pSB4C5. Samples are following

Our samples are following four.

  • target B0034+ anti-sense B0034
  • target B0034+ anti-sense B0032
  • target B0032+ anti-sense B0034
  • target B0032+ anti-sense B0032

We examined each samples with And without IPTG induction.

Methods for assay

We selected mRFP as a 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. Cultivated the colony in 4 mL LB culture for about 22 hours
  2. Controled turbidity up to 0.1 at OD600
  3. cultivated the colony in 2 mL M9ZB culture for 30 hours (IPTG induces antisense RNA, add 20 uL)
  4. Measured fluorescence after 30 hours
Fig. 12 Value of fluorescence of IPTG with and without each sample
Fig. 13 Rate of IPTG(+) and IPTG(-) Numeric become small by inducing anti-sense RNA with IPTG. A vertical axis numeric from 100 leaves efficiency of suppression.
Fig. 14 Quantity of anti-sense RNA with IPTG and without. We have no date without IPTG.

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 worked.

Discussion

  1. Anti-sense was not induced by IPTG; it is leaking.
    2. Seen from Fig. 12, 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. 10, it was confirmed that asB0032 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
    3. Although it would be a contrasting discussion to discussion 1, from fig.10, we could not find little gap in fluorescence of mRFP in antisense B0032 with/without IPTG inducing. Likewise from fig.12, 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 confirm 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
    4. Seeing Fig.10 and 12, 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
    2. 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
    3. 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
    4. 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.

Fig. 15 Rate of RBS used BioBrick parts

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.



Reference

  1. N. Nakashima et al. (2006) Paired termini stabilize antisense RNAs and enhance conditional gene silencing in Escherichia coli. Nucleic Acids Res 34: 20 e138
  2. N. Nakashima and T. Tamura (2009) Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli. Nucleic Acids Res 37: 15 e103

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