Team:ShanghaiTech/Project Oribosome
Orthogonal ribosome
Orthogonal ribosomes are a kind of ribosomes that have been engineered and reprogrammed. In our project, we mainly focus on the binding interaction between mRNA and the 16S ribosomal RNA (16S rRNA), hoping to create an isolate translation system. In the phase of translation, the small subunit of ribosome bind to the RBS(ribosome binding site) on the mRNA to start translation. While the altered 16S rRNA can only recognize and bind to an altered Shine-Dalgarno sequence, which then initiates the translation of the downstream gene.
Since the 16S rRNA and the SD sequence is mutated and different from the endogenous system, the orthogonal ribosome’s control of translation is separated from the host’s.
Key information
The characteristic of the orthogonal ribosome, including feasibility, orthogonality and compatibility with the host's ribosome.
Through desktop research, we found several different editions of orthogonal ribosomes. After consideration, we chose four pairs of orthogonal ribosomes and orthogonal RBS(ribosome binding site) to test and try to find the best to use in our system. They are labeled as A2, B8, C9 from Jason Chin's lab and TJU TJU_2012 iGEM team. Although no constructed plasmid was available from Jason’s or TJU, the way they mutated the host ribosome 16s rRNA were provided in the paper they published. Therefore, we are luckily to have the chance to construct these orthogonal ribosomes by mutagenesis in our own lab.
The construction of orthogonal ribosomes
As the ribosome binds to the mRNA only depending on the small subunit 16s rRNA, we only need to construct orthogonal 16S rRNA. To obtain the orthogonal 16s rRNA, we first cloned E.coli genomic DNA 16s rRNA rrnB to T-Vector. According to the sequence provided in the paper or website, we mutated the Shine-Dalgarno site for every version of orthogonal ribosome. And for A2, B8, C9 orthogonal 16s rRNA from Jason Chin's lab, an extra mutation was performed from 722 to 723 bp of rrnB. The extra mutation helps the ribosome to form a bulge proximal to the minor groove of the SD helix formed between the ribosome and mRNA, which increase the orthogonal ribosome’s orthogonality.
Fig.1 The library of orthogonal ribosome - orthogonal RBS pairs.
Orthogonal ribosome characterization experiments
We develop two kinds of circuits to characterize the orthogonal ribosome.Specifically, the host's ribosome or o-ribosome pool utilised for translation is controlled by the selection of ribosome binding site (RBS).
For the first kind of circuits, there is only one reporter gene GFP on it.
Fig.2 GFP under orthogonal RBS within or without J23100- orthogonal 16S rRNA (UP: Plasmid A; DOWN: Plasmid B)
A. We characterize orthogonal ribosome system in terms of GFP expression under the orthogonal RBS(ribosome binding site) in the absence or presence of corresponding orthogonal 16S rRNA to characterize its orthogonality to the host genome.
| orthogonal 16s rRNA | orthogonal RBS |
|---|---|
| A2 | A2 |
| C9 | C9 |
| TJU | TJU |
B. We characterize orthogonal ribosome system in terms of GFP expression under orthogonal RBS in the presence of different non-corresponding orthogonal 16S rRNA to characterize its orthogonality with each other.
| orthogonal 16s rRNA | orthogonal RBS |
|---|---|
| A2 | B8 |
| A2 | C9 |
| C9 | A2 |
| C9 | B8 |
| TJU | A2 |
| TJU | B8 |
| TJU | C9 |
For the second kind of circuits, there is only two reporter genes on it. One is RFP with degradation LVA tag under host RBS, the other is GFP with degradation LVA tag under orthogonal RBS.
Fig.3 GFP is under orthogonal RBS while RFP is under host RBS (UP: Plasmid A; DOWN: Plasmid B).
| orthogonal 16s rRNA | orthogonal RBS |
|---|---|
| C9 | C9 |
| TJU | TJU |
We developed two new circuits both consists of two parts: the original RFP pool and an orthogonal GFP pool. Genes encoding RFP is supposed to express as the host's 16s rRNA always exists, while the expression of GFP mainly depends on the presence of orthogonal 16s rRNA.
*For characterisation experiments, transformed cells were grown from stocks overnight, and subcultured for three to four hours, the cultures were diluted to 0.05 O.D. and aliquoted into a 96 wells plate or cell culture tube.
Results
The construction of orthogonal ribosomes
The orthogonal ribosome
The plasmids contains reporter gene GFP or RFP under the orthogonal ribosome is directly synthesized by the third-party company.
Experiments were carried out by culturing 10ul cells in 2ml LB, and absorbance value (OD 600) and fluorescence signal of GFP and RFP were monitered over time using a microplate reader. Fluorescence values were divided by absorbance values to give a series of normalised data. Both the original and normalised data were presented below.
Experimental results showed that Jason's orthogonal pool sufficiently controlled the expression of GFP. The fluorescence signal of GFP was nearly close to zero without orthogonal 16S rRNA, but when the orthogonal 16S rRNA existed, the fluorescence signal increase over time. Besides, the fluorescence signal of RFP increased with time as well, no matter whether the o-16S rRNA existed.
Fig.4 A) Cells contain plasmids with TJU's orthogonal pool and oRBS grew well and reached the plateau after 16 hours. B) Cells contain plasmids with Jason's orthogonal pool and oRBS grew well and reached the plateau after 15 hours.C) Cells contain plasmids with Jason's orthogonal pool, oRBS and o16S rRNA grew well and reached the plateau after 13 hours.
Fig.5 D) Cells contain plasmids with TJU's orthogonal pool and oRBS hardly expressed GFP without o-16S rRNA, while RFP expressed well as the host's 16S rRNA existed. E) Cells contain plasmids with Jason's orthogonal pool and oRBS hardly expressed GFP without o-16S rRNA, while RFP expressed well as the host's 16S rRNA existed. F) Cells contain plasmids with Jason's orthogonal pool, oRBS and o16S rRNA expressed GFP and RFP well.
Fig.6 G) The expression level of GFP in two kinds of cells contain plasmids under the control of Jason's orthogonal pool, only differ in whether has genes encode o16S rRNA. H) The expression level of RFP in two kinds of cells contain plasmids under the control of Jason's orthogonal pool, only differ in whether has genes encode o16S rRNA.
References
Alexander P. S. Darlington, Juhyun Kim, José I. Jiménez, Declan G. Bates. (2018) Dynamic allocation of orthogonal ribosomes facilitates uncoupling of co-expressed genes. Nature Communications 9:1.