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

The targets of these asRNA 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 stemmed anti-sense complex.
Fig. 6 Blue; anti-sense 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 a colony of transformed bacteria in 2 ml of LB medium (until the turbidity at OD600 reached 0.1).
  2. Retrieved the bacteria and cultivated them in 2 ml of M9ZB medium
  3. Centrifuged the culture at 10,000 rpm / for 2 min / at 25°C
  4. Removed the supernatant and add M9ZB medium then voltex the pelet.
  5. Performed RT-PCR
  6. Measured absorbance of 260 nm about cDNA.
Fig. 9 Anti-sense B0034 is induced by IPTG

Results

We assessed the efficiency to suppress expression of two kinds of asRNA family: one has Paired-Termini structure (Nakashima's stem) and pHN1257 as the vector, the other has newly designed H-stem structure. For each kind of asRNA, we prepared 4 types of E. coli:

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

We examined each sample with / without IPTG induction.

Nakashima's stem

We assessed whether asRNA with Nakashima's stem works. We used Nakashima’s plasmid (pHN1257[1][2]) as a vector. We double transformed separate plasmids of anti-sense and target gene and had an assay. All anti-sense constructs are on pHN1257 and all target constructs are on pSB6A1. The sample E. coli were cultivated for 18 h in M9ZB medium.

Details of pHN1257 vector

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

Fig. 10 Fluorescence strength of each sample with / without IPTG
Fig. 11 Fluorescence strength ratio for IPTG(+) / IPTG(-). smaller values mean that the target gene was suppressed by IPTG induction.

Fig. 11 shows the fluorescence strength ration between IPTG induction (+) / (-). Smaller values indicate that the mRFP gene was suppressed because of asRNA induced by IPTG. In the case of B0034, both asB0034 and asB0032 suppressed the target gene expression. However, for B0032, neither asB0034 nor asB0032 was confirmed to work. From these results, we were not able to confirm specificity of asB0034, but toward the construct using B0034, asB0034 down-regulated the expression by 40%, and asB0032 did by 80%. In Nakashima's results, the efficiency of down-regulation was 78%, so we got the same result.

H-stem

We inserted anti-sense between H-stem instead of PT structure and assayed their efficiency.

Fig. 12 Fluorescence strength of each sample with / without IPTG
Fig. 13 Fluorescence strength ratio for IPTG(+) / IPTG(-). smaller values mean that the target gene was suppressed by IPTG induction.
Fig. 14 Quantity of anti-sense RNA expressed with and without IPTG induction. We have no data without IPTG.

From these experiments, we were not able to confirm whether asRNA worked 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 data of IPTG-.) From this result, we confirmed that, at least, asB0034 was expressed.

Discussion

We are assuming three possible causes of that H-stem system made no difference between the cases with and without IPTG induction.

  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- for any sample. Since fluorescence showed no difference, it could be assumed that the asRNA was constantly over-expressed regardless of IPTG induction. Also, on Fig. 10, it was confirmed that asB0032 works on B0034 construct, but it was not functional on H-stem system. 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. Anti-sense was not expressed sufficiently.
    3. From fig.10, we found little gap in fluorescence of mRFP for constructs using B0032 with/without IPTG induction. Likewise from fig.12, we found little gap either. In consideration of these facts, we guessed that asRNA expression were not sufficient. We confirmed the existence of antisense B0034 by sequencing the vector, though we did not confirm about antisense B0032. The reason is the difficulty to sequencing of DNA which has stem-loop structures.

  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 we cultured several colonies derived from same origin of plasmids. However, the expression of mRFP showed gap between colonies despite of same level of turbidity. Therefore, because of changes of copy number of target gene, expression of target genes weren’t consistent, and then we found that it was difficult to measure and estimate the activation of anti-sense by fluorescence.

Improvement

  1. Analysis by RT-PCR
    2. By analyzing quantity of asRNA, we showed the expression can be induced using IPTG. We will anti-sense works even if copy number is unstable by comparing mRNA of target gene with / without anti-sense.

  2. Improvement of medium to induce anti-sense by adding IPTG easily
    3. To induce asRNA by IPTG easily, we have used M9ZB medium. However the medium includes glucose, IPTG induction may be too late. We will try to use LB medium to enable IPTG induction.

  3. Stability of copy number in target gene
    4. It was published that low copy plasmid often occurs movement of copy number. We need to select plasmid with higher copy number. We will use medium included an 1.5 times antibiotic for screening.

We are going to show positive result in Boston!

Conclusion

We showed asRNA works in case using Nakashima’s stem. On the other hand, in case of H-stem system, we could confirm only transcription of anti-sense but we could not get a proof that anti-sense worked. We want to show some results in the presentation at Boston by re-thinking copy number of plasmids or medium for assay to work anti-sense 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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