• Introduction
• Method
• Evaluation
• Future work

RNA Scaffold

Introduction

Background

SplitGFP
Split GFP is split into two fragments between amino acid residue 158 and 159, FA (N-terminal domain) and FB (C-terminal domain). FA alone can’t emit fluorescence. Equally, FB alone can’t emit fluorescence. When FA and FB co-express and meet, the fragments associate and restore fluorescence. Split GFP is used to research RNA detection and localization in vivo by fusion with RNA binding proteins.

Aptamer
Aptamers are nucleic acid or peptide molecules, bind to a specific target molecule. There are many target molecules, for example enzyme, acceptor, virus protein, organic molecule, and so on. RNA aptamer requires a secondary structure formation (hairpin-loop, G-quartet, pseudo knot and bulge loop) to bind to a specific target molecule. RNA aptamers are useful in bio-engineering, because they are easier than proteins for mutation and prediction of secondary structure. In addition, RNA aptamers don’t need to be translated and they work rapidly.

MS2 and PP7
MS2 and PP7 are one of the single strand RNA phage coat proteins. RNA aptamer sequences and structures to recognize MS2 and PP7 coat protein have been determined.

Previous work
Camille J. Delebecque and his colleagues have reported organization of bacterial metabolism in vivo by using an RNA scaffold1. They used PP7 and MS2 fused with split GFP to evaluate the scaffold, consists of PP7 and MS2 aptamer domains, whether PP7 and MS2 proteins assemble. They succeed in assemble PP7 and MS2 proteins.
2012 ZJU-China team registered parts (BB_K73800, BB_K73804, BB_K73805), PP7 and MS2 fused with split GFP which bind to PP7 and MS2 aptamer domains respectively. FA and FB emit fluorescence when they meet. Therefore, when a scaffold containing PP7 and MS2 aptamer domains are expressed, FA and FB come closer and emit fluorescence due to two fusion protein bind to each aptamers.

Objective

Functionalization of protein using RNA without translation
Tokyo-NoKoGen decided to use these parts to detect an RNA fragment that is produced when HHR causes self-cleavage. There is a complementary sequence to RNA scaffold in HHR, therefore one aptamer does not form secondary structure alone. Only when HHR causes self-cleavage and release the scaffold, the scaffold can form both aptamer domains and result in emitting fluorescence (Fig. 1). On the other hand, when HHR doesn’t cause self-cleavage and release the scaffold, the scaffold can’t form one aptamer domains and result in no fluorescence.
So we will regulate GFP fluorescence by RNA, and the response is faster than protein regulation because it doesn’t need to translate. In addition, we can regulate automatically to use RNA oscillation based on HHR.

Fig. 1 How is fluorescence emitted when HHR causes self-cleavage.

Method

Construction of Parts
Ⅰ Construction of split GFP fused with MS2 and PP7 proteins
Ⅱ Construction of RNA scaffold
Ⅲ Construction of split GFP + RNA scaffold

Ⅰ Construction of split GFP fused with MS2 and PP7 proteins
1) We used synthesized sequences of split GFP fused with MS2 (FA-linker-MS2) and split GFP fused with PP7 (FB-linker-PP7) respectively, reference of BioBrick part BBa_K738004 (FA-2X-MS2) and BBa_K738005 (FB-2X-PP7) (ZJU-China 2012). Overlap extension PCR was performed to connect the synthesized fragments.

2-1) The sequence of FB-linker-PP7 was digested at EcoRI and SpeI sites, and BBa_B0015 (double terminator) was digested at EcoRI and XbaI sites, followed by ligation (Fig.2). The region of FB-linker-PP7 and double terminator was amplified by PCR using primers; forward primer has XbaI site and RBS. pSB1A3 and the assembled sequence was digested with XbaI and SpeI (Fig.3).

Fig.2 Assemble of double terminator downstream of FB-linker-PP7

Fig.3 Assemble of RBS upstream of FB-linker-PP7

2-2) The region of constitutive promoter and RBS in BBa_K317026 (Tokyo-NoKoGen 2010) was amplified by PCR using two primers. The sequence for FA-linker-MS2 constructed in 1) was amplified by PCR. PCR products were ligated into pSB1C3 vector (Fig.4).

Fig.4 Assemble of constitutive promoter and RBS upstream of FA-linker-MS2

3) The parts constructed in 2-1, 2-2) were assembled by using digestion with a restriction enzyme. This fragment was inserted into pSB1A3 vector, followed by transformation in E. coli (DH5α) (Fig.5).

Fig.5 Construction of split GFP fused MS2/PP7 proteins

Ⅱ Construction of RNA scaffold
4) BBa_K1053111 (Tokyo-NoKoGen 2013) for RNA scaffold-DT containing MS2 and PP7 aptamer domains, and we chose BBa_J23100 (Berkeley 2006) for constitutive-high promoter as its promoter.
5) The former plasmid was digested at XbaI and PstI site, the later plasmid was digested at SpeI and PstI sites. The digested parts were ligated together. It is our new BioBrick part, BBa_K1053110 (Fig.6). After this part was inserted in pSB1A3, it was transformed in E. coli (DH5α).

Fig.6 Construction of RNA scaffold

Ⅲ Construction of split GFP + RNA scaffold
Each parts constructed in 3) and 5) was digested and ligated (Fig.7), followed by transformation in E. coli (DH5α).

Fig.7 Assemble of split GFP and RAN scaffold

Evaluation

Three transformants (shown below in Fig.10) were inoculated from in 3 mL LB medium with relative resistances, and incubated in 37 °C at 150 rpm. After the pre-cultured for over 12 h, the main cultivation was done in 3 mL LB medium with relative resistances inside a L form tube. The experiments were started at cell density at 600 nm (OD600) of about 0.1. The GFP fluorescence intensity (FI) and cell density (OD600) were taken, periodically at 2 h intervals for 12 h by Plate Reader.

Fig.8 Evaluation of the FI/OD600 on split GFP and RNA scaffold in E. coli

Fig.10 Three transfomants; (A)only RNA scaffold, (B)only split GFP fused MS2/PP7 proteins
(C)RNA scaffold + split GFP.

Result

coming soon…


Fig.9 Comparison of normalized the GFP fluorescence intensity to cell density (FI/OD600).

Discussion

Future work

We are going to construct the HHR fused with RNA scaffold (HHR-RNA scaffold) and no self-cleavage mutant (HHR*-RNA scaffold). HHR-RNA scaffold is unable to form RNA scaffold without self-cleavage because RNA scaffold contain complementary sequence to HHR. So we will evaluate whether the formation of RNA scaffold is regulated by self-cleavage or not. After that, we will construct RNA oscillation system based on HHR that periodically release an RNA fragment containing RNA scaffold and result in engineering twinkle E. coli. We will try to make Split luciferase and fuse the split luciferase to MS2 and PP7. By using the Split luciferase, E. coli emit light periodically like a firefly.
The oscillation system can also use its RNA scaffold for enzyme reactions. We sought other aptamers to increase the variety of BioBrick for enzyme reactions. Hung-Wei Yiu has reported RNA detection in living bacterial cells caused by binding of two RNA binding peptide, HTLV-1 Rex peptide and λN peptide, to two RNA aptamer. They fused these two peptides to split GFP and detect RNA which contains HTLV-1 Rex peptide and λN peptide aptamer. So we will construct and evaluate the system and resister as a BioBrick.



Reference
[1] Natalia E. Broude, “Analysis of RNA localization and metabolism in single live bacterial cells: achievements and challenges” Molecular Microbiology (2011) 80(5), 1137-1147

[2]Delebecque, C.J., Lindner, A.B., Silver, P.A. & Aldaye, F.A., “Organization of Intracellular Reactions with Rationally Designed RNA Assemblies” Science 333, 470-474 (2011).

[3] Hung-Wei Yiu, Vadim V. Demidov, Paul Toran, Charles R. Cantor and Natalia E. Broude, "RNA Detection in Live Bacterial Cells Using Fluorescent Protein Complementation Triggered by Interaction of Two RNA Aptamers with Two RNA-Binding Peptides" Pharmaceuticals 2011, 4, 494-508

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