• overview
• modeling
• future work

Making RNA Oscillator
Introduction Background
Protein oscillator
(There are many kinds of gene regulation system. One of them is repressor system that is used...)
The oscillating network was built by using three transcriptional repressor systems in which the first repressor protein suppresses the expression of second repressor gene, whose protein suppresses the transcription of the third gene, and the protein the third gene code suppresses the first gene expression.

Hummer headed ribozyme

Hummerheaded ribozymes (HHRs) are found in many species and known to have a capability to self-cleave. Recently, engineered HHRs responding to trans-acting RNAs (taRNAs) in E.coli were reported. In absence of taRNA, the HHRs can self-cleave, but in presence of taRNA, their capability of self-cleavage is inhibited due to hybridize with taRNA and change their structures.


We developed the new oscillator system that is composed only RNAs not using repressor gene and protein. We created three taRNA connected hummerheaded ribozymes (HHR-taRNA) that sense to another taRNA. The same way as the protein oscillator, three HHR-taRNAs inhibits other one’s self-cleavage such as three-way standoff. When the first HHR-taRNA is self-cleaved, connected the first taRNA that binds to the second HHR-taRNA is free and suppresses the second HHR-taRNA self-cleavage. When second HHR-taRNA is self-cleaved, the second taRNA that binds to the third HHR-taRNA is free and suppresses the third HHR-taRNA. Finally, when third HHR-taRNA is self-cleaved, the third taRNA that binds to the first HHR-taRNA is free and suppresses the first HHR-taRNA, completing cycle.


Object
Construction of Three of orthogonal taRNA generator which repressed by another taRNA.
taRNA responsive HHR (TR-HHR) was fused with taRNA. By using orthogonal riboregulator sequences, we designed three of taRNA generators.


Design
TR-HHR was designed by Benedikt Klauser and Jo¨ rg S. Hartig. Their TR-HHR were inspired by the riboregulatory system previously reported by Collins and coworkers. They made use of sequence domains of the riboregulator RR12. And, orthogonal variants of riboregulator RR12 (RR12y, RR42) were reported by Collins and coworkers. So, by using the sequences of these orthogonal riboregulators, we designed three orthogonal TR-HHRs.


図１


Method
1. Construction of TR-HHR
2. Construction of TR-HHR variants
3. Construction of taRNA variants
4. Construction of TR-HHR-taRNA



1. Construction of TR-HHR
We used the TR-HHR developed by Klauser et al.[1] Two overlap primers designed, and PCR was performed to connect them. The PCR product was ligated into pSB1C3 vector. 図


2．Construction of TR-HHR variants


By inverse PCR using pSB1C3-TR-HHR as a template, we could obtain three TR-HHRs, TR(12)-HHR, TR(12y)-HHR, and TR(42)-HHR. 図


3．Construction of taRNA variants


By inverse PCR using pSB1A3-taR12-DT as a template, we could obtain three taRNAs, taR12, taR12y, taR42. 図


4．Construction of TR-HHR-taRNA


TR-HHR-taRNAs were constructed by overlap PCR.


図 Evaluation


I. Evaluation of TR-HHR


II. Evaluation of TR-HHR-taRNA


I.　Evaluation of TR-HHR

In order to evaluate the regulation of HHR’s self-cleavage activity by taRNA, GFP gene was connected with the HHR or inactive HHR (*HHR), which has a mutation lose the activity. These fragments were subcloned into conventional biobrick standard vector pSB3C5.
1) E. coli TOP10 strain was co-transformed by two plasmids listed bellow,

A) pSB1A3-taR12-DT

pSB3C5-TR(12)-HHR -GFP-DT

B) pSB1A3-empty

pSB3C5- TR(12)-HHR -GFP-DT

C) pSB1A3-taR12-DT

pSB3C5-TR(12)- * HHR-GFP-DT

D) pSB1A3 empty

pSB3C5- TR(12)- * HHR -GFP-DT

E) pSB1A3-empty

pSB3C5-empty

2) Bacterial cultures were incubated in LB medium at 140 rpm and 37℃ for 12 hour.
3) Then, cultures was diluted to an OD595 of 0.0125 and induced with or without 0.1% arabinose.
4) Cultures were incubated with shaking at 140 rpm and 37℃ for 8 hours in 1 mL of LB medium.
5) OD595 and GFP fluorescence intensity were measured.
All experiments were performed using three cultures per each sample.



[評価の図]

II. Evaluation of TR-HHR-taRNA

We examined whether TR-HHR-taRNA’s output can be repressed by corresponding taRNA.
1) The plasmids as shown below was used to transform E. coli Top10.
A) pSB1A3-Pbad -TR(42)HHR-taR12-DT
pSB3C5-crR12-GFP


B) pSB1A3-Pconst(H)-taR42-Pbad-TR(42)HHR-taR12-DT
pSB3C5-crR12-GFP-DT


C) pSB1A3-Pbad-taR12-DT

pSB3C5-crR12-GFP
D) pSB1A3-empty
pSB3C5-crR12-GFP


E) pSB1A3-empty
pSB3C5-empty


2) The transformants were pre-cultured in LB medium at 37 degrees celcius for 8 hours.
3) Then, cultures was diluted to an OD595 of 0.0125 and induced with or without 0.1% arabinose.
4) Cultures were incubated with shaking at 140 rpm and 37℃ for 8 hours in 1 mL of LB medium.
5) OD595 and GFP fluorescence intensity were measured.


[評価の図]
・Result
・Discussion
・Future work
・Reference
  Object Making RNA Osciilator Construction of taRNA generator which repressed by another taRNA. taRNA responsive HHR(TR-HHR) was fused with taRNA. By using orthogonal riboregulator sequences, we designed three of taRNA generators. I) 1) Construction of pSB1C3-TR-HHR(RBS) We used the TR-HHR developed by Klauser et al.[1] Two overlap primers designed, and PCR was performed to connect them. The PCR product was ligated into pSB1C3 vector. Evaluation of TR-HHR II) Construction of the plasmids to evaluate TR-HHR 1) Construction of pSB1C3-PBAD-TR-HHR(RBS)-GFPuv-DT PBAD was digested and ligated into pSB1C3-TR-HHR(RBS). GFPuv was digested and ligated into DT. Then, GFPuv-DT was digested with and ligated into pSB1C3-PBAD-TR-HHR(RBS). Evaluation of TR(42)-HHR-taR12 ・pSB1A3-Pbad -TR(42)HHR-taR12-DT ・pSB3C5-crR12-GFP ・pSB1A3-Pconst(H)-taR42-Pbad-TR(42)HHR-taR12-DT ・pSB3C5-crR12-GFP-DT ・ pSB1A3-Pbad-taR12-DT ・ pSB3C5-crR12-GFP (positive control) ・pSB1A3-empty ・pSB3C5-crR12-GFP (negative control) ・pSB1A3-empty ・pSB3C5-empty

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