This year, the theme of iGEM Gifu is to synthesize a long chain protein with circular mRNA. It is a sequel to our theme of the last year.
Here, we explain our theme of the last year.
The circular mRNA is expressed in E. coli by using a mechanism of self-splicing.
In td gene of T4 phage, there is the group 1 intron. A part of the RNA transcribed from the group 1 intron is a ribozyme which catalyzes a splicing reaction, and this RNA splices itself.
The circular mRNA can be expressed in E. coli by cloning a domain which functions as ribozyme for splicing of the td gene, and putting it in the plasmid. This method is called “PIE method.”
In last year, we succeeded in designing the sequence which synthesizes circular mRNA and long chain protein in Escherichia coli. In this year, we had 2 purposes in our study.
One was an efficiency of the circularization. The efficiency was lower in our previous study. This was why the splicing was hard to happen because two sequences to act as ribozyme for splicing were far each other. So we incorporated complementary sequences around the ribozyme regions. We thought that the treatment brought two regions close and the efficiency of the circularization improved.
The other was to synthesize useful proteins. In our previous study, synthesized long-chain proteins lose their function because the folding of the proteins was broken. So we incorporated linker sequences into circular mRNA to synthesize the functional long-chain protein.
The group I intron in td gene of T4 phage has self-splicing mechanism. The self-splicing is a mechanism that circularizes the intron and connects exons. This is catalyzed by several base sequences of the ends of the introns as a ribozyme. We permuted exons and introns with the mechanism and attempted an exon circularization. So we constructed mRNA circularization devices in last year.
We explain the circularization mechanism of group I intron with td gene of T4 phage as an example. Td gene consists of an upstream exon, an upstream intron, an ORF, a downstream intron and a downstream exon. This mechanism is divided into 3 steps. As the first step, a nucleophilic attack by a guanosine separates the upstream exon from the upstream intron and then the guanosine bonds to the 5’ end of the upstream intron.
As the second step, the downstream exon is separated from the downstream intron by a nucleophilic attack. The nucleophilic attack takes place by a hydroxy group at the 3’ end of the upstream exon. (Figure 1)
Fig.1 Self-splicing in T4 phage: the first and second step (Blue: intron, Orange: exon)
As the third step, the upstream intron bonds to the downstream intron by an attack on an adenine of the upstream intron. The attack takes place by a hydroxyl group of an end of the downstream intron. And then a circular intron is formed.(Figure 2)
Fig.2 Self-splicing in T4 phage: the third step (Blue: intron, Orange: exon)
The permuted intron-exon method : PIE Method
We can use the group I intron self-splicing mechanism in td gene of T4 phage to circularize mRNA. The group I intron self-splicing is a mechanism that circularizes an intron and connects exons. It occurs after transcription. The self-splicing is catalyzed by several base sequences of the ends of introns as a ribozyme. We permuted exons and introns with the mechanism and attempted an exon circularization. So we constructed mRNA circularization devices. We induced a protein coding sequence and them into E. coli. We created circular mRNA and synthesized massive long-chain protein with it.
Fig.3 Method of the synthesis of the long chain protein
Two exons are connected with each other in the circularization system; furthermore an exon can theoretically be circularized by the system. (Fig.4)
Fig.4 An idea of mRNA circularization (Blue: intron, Orange: exon)
The PIE method is shown below(Figure 5). First, we picked out the intron and splice sites in the exon from td gene in T4 phage. Second, sandwich the sequence that you want to circularize between preceding fragments.
Fig.5 PIE method
Last year, we confirmed that making mRNA circular in E. coli by PIE method and synthesizing long-chain protein. (2014 Team Gifu)
Qualitative test of circular mRNA was conducted by using exonuclease.
Exonuclease - enzyme which cleaves mRNA from the end-cannot cleave circular mRNA, because it doesn’t have end site.
We perform RNA extraction, exonuclease processing, RT-PCR.
We can confirm bands by electrophoresis PCR production even if circular mRNA does
In addition, we found that cyclic mRNA made because it was revealed that its sequence not to appear when a reaction was not caused (sequence including the joint) circulization when we read the sequence of this band.
Fig.6 RT-PCR of RNA which carried out each nuclease processing
Synthesize-long chain protein
Qualitative test of long-chain protein was conducted by using SDS-PAGE.
We found huge protein by SDS-PAGE when crushing E. coli had circular mRNA.
In addition, we found that the protein was derived from RFP by Western blot.
Thus, we concluded circular mRNA made long-chain protein.
Fig.7 SDS-PAGE of RFP produced in E. coli
Fig.8 Western blotting
Fig.9 The protein has the fluorescence, or not.
Fig.10 Efficiency of circularization
We calculated the efficiency of the circularization by using MPN method. As the result, the efficiency was only 2.5%.
Also, long chain RFP synthesized by circular mRNA showed no fluorescence.