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Latest revision as of 00:34, 18 October 2018

MiniToe, a cis-regulatory RNA element

The background

In type I and type III CRISPR systems, the Cas6 endoribonuclease splits the specific pre-crRNAs in a sequence-specific way to generate 60-nucleotide (nt) crRNA products in which segments of the repeat sequence flank the spacer (to target "foreign" nucleic acid sequence) [1] The pre-crRNA target site adopts a stem-loop structure (the specific 22nt RNA hairpin) with five base pairs in A-form helical stem capped by GUAUA loop containing a sheared G11-A15 base pair and a bulged nucleotide U14. In the binding structure of Csy4-RNA complex, the RNA stem-loop straddles the β-hairpin formed by strands β6-7 of Csy4[2]. And once the Csy4/RNA complex formed, the structure will stay stable and hard to separate.

We find that Csy4/RNA complex is a good material to design a new tool based on post-transcriptional regulation. And the stem-loop structure (the specific 22nt RNA hairpin) is used to design the tool.

[http://2018.igem.org/Team:OUC-China/Design Click here to see the details of tool we designed!]

[1].Przybilski R, Richter C, Gristwood T, et al. Csy4 is responsible for CRISPR RNA processing in Pectobacterium atrosepticum.[J]. Rna Biology, 2011, 8(3):517-528.
[2].Haurwitz R E, Jinek M, Wiedenheft B, et al. Sequence- and structure-specific RNA processing by a CRISPR endonuclease[J]. Science, 2010, 329(5997):1355-1358.

The structure of miniToe

This year, we design a new tool based on Csy4 and hairpin we mention before. One of the key role in tool is the cis-regulatory RNA element named miniToe which can be recognized by Csy4. On the level of DNA, we named the gene miniToe part. And on the level of RNA, we focus on the miniToe structure.

The translational control module named miniToe is constructed by inserting a Csy4 recognition site between a RBS and cis-repressive RNA element, which can be specifically cleaved upon Csy4 expression. The whole system including miniToe and Csy4 compose a kind of tool at the level of post-transcription.

The tool with three modular parts:

• A cis-repressive RNA served as a translational suppressor by pairing with RBS as the critical part of miniToe structure.
• A Csy4 site as a linker between cis-repressive RNA and RBS, which can be specifically cleaved upon Csy4 function.
• A CRISPR endoribonuclease Csy4.

Fig.1 The structure of miniToe on the level of RNA.

In the project, the superfolder green fluorescent protein (sfGFP) is the reporter gene to reflect output of our system under miniToe regulation, the expression of this gene is driven by a constitutive promoter. The stability of miniToe structure is crucial. Hence, before wet experiment, we predicted the structure of full-length transcript of this circuit as well as miniToe structure.

Fig.2 The structural prediction of the circuit and miniToe.

The function of miniToe

To verify the feasibility and function of miniToe, the following circuits were designed for testing the function of miniToe. An inducible promoter P tac controls the expression level of Csy4. The cis-repressive RNA coding sequence is inserted at the upstream of reporter (sfGFP) gene，which is controlled by a constitutive promoter from Anderson family named J23119.

Fig.3 The two plasmids of miniToe test system.

Without Csy4,the cis-repressive RNA pairs with RBS very well, so the switch just turns off which means that no protein will be produced. Otherwise, with the presence of Csy4, the translation turns on. In this way, the expression of downsteam gene can be regulated.

Fig.4 The mechanism of miniToe system.

The aim of miniToe family

We design miniToe family to meet the aim, "One system, diverse expression". That means by using one system we can even achieve flexable expression of target gene. The miniToe-WT which is a wild type hairpin is one of the miniToe family. And then we use sfGFP as our reporter.( miniToe test system-1, miniToe test system-2, miniToe test system-3, miniToe test system-4, and miniToe test system-5.)

By quantitatively adjusting the miniToe hairpin, we can even achieve a stoichiometry expression of the target proteins in a polycistron gene expression construct. In our design, the duplex can be modularized, the mutants of the crRNA may potentially fine-tune the translational activator. Thus offering diversity to future design and integration into complex genetic circuits.

Result

Growth rate measurement

As preliminary experiment, the growth rate measurements are essential. The curve below demonstrates all the groups have almost the same tendency of OD600 with the negative control strain during the entire cultivation period. It means that miniToe system has no negative influence on the growth of recombinant strain. The metabolic stress by two plasmids is not harmful to the recombinant strains.

Fig.5 Growth curve of strains we used in experiments. Error bars represent standard deviation of four biological replicates. (Measured by microplate reader)

Proof of functions

1. By microplate reader

The microplate reader is used to test the fluorescence intensity of superfolder green fluorescent protein (sfGFP) which is changed over time. The aim is to prove that miniToe system can control the downstream gene expression during the whole cultivation period.

The following chart shows the dynamic curve measured by microplate reader every two hours. The yellow line refers to the test group which is recombinant strain (with the whole miniToe system) with IPTG (0.125mM). The blue line shows the change of fluorescence intensity in recombinant strain (with the whole miniToe system) without IPTG (0mM). The green line refers to another control group which only has pReporter without the pCsy4 in strain. The results help us to prove two problems in miniToe system.

Fig.6 The fluorescence intensity of sfGFP by microplate reader during the entire cultivation period. There are three groups. The yellow line refers to a test group with IPTG (0.125mM). The blue line refers to a group without IPTG (0mM). The green line refers to a control group only with pReporter. Error bars represent standard deviation of three biological replicates(Measured by microplate reader).

The first problem is whether miniToe structure can fold exactly on the level of RNA in reality. In Fig.6, a control group (the green line) is relatively stable during the whole process comparing with other two control groups. It means the miniToe structure without Csy4 folds well on the level of RNA and also keep OFF state so the changes of fluorescence intensity cannot be detected.

The second problem need to prove is whether miniToe system can work successfully as a switch to regulate the downstream genes. Obviously, in the Fig.6, there is a rise in expression of sfGFP between two lines in the whole process. The yellow line is the test group with the IPTG and the blue line is a control group without IPTG. It is not difficult to find that the fluorescence intensity of control group (the blue line) is always lower than test group (the yellow line). These data strongly support that the increased expression of the target gene sfGFP is indeed due to cleavage of Csy4 site that exposed the RBS to restore translation. It means miniToe system can work successfully.

Fig.7 The result from other four teams which proved our conclusions. Error bars represent standard deviation of four biological replicates.

2. By flow cytometric

We also tested miniToe system by flow cytometric. In Fig.7, it's easy to distinguish the two groups (blue & white) and the test group (+IPTG) has the obvious increase compared with the control group (-IPTG). The result shows the same conclusions mentioned before.

Fig.8 Flow cytometric measurement of fluorescence of sfGFP. Histograms show distribution of fluorescence in samples with test group with IPTG (green) and control group without IPTG (white). Crosscolumn number shows fold increase of sfGFP fluorescence. The test group is a recombinant strain (with the whole miniToe system including two plasmids) with IPTG (0.125mM). And the control group is a recombinant strain (with the whole miniToe system including two plasmids) without IPTG (0 mM).

3. By Fluorescent Stereo Microscope Leica M165 FC

The sfGFP accumulated during the cultivation period so the fluorescence can be observed by microscope after 8 hours. The fluorescent can be seen by naked eyes though microscope!

Fig.9 The expression of sfGFP by Csy4-WT&miniToe. The plotting scale is on the right corner of images. The images on the left shows E. coli without fluorescence excitation. The images on the right represent situation when fluorescence excitation.

MiniToe family

We also design some miniToe mutants by changing the sequences of Csy4 hairpin. All the miniToe mutants we redesign form a samll library. The aim is to obtain different hairpins which have various activity for Csy4 recognition and RNA cleavage.

[http://2018.igem.org/Team:OUC-China/Design You can get more information about miniToe mutants here!]

Here are the parts we submit! miniToe test system-1, miniToe test system-2, miniToe test system-3, miniToe test system-4, and miniToe test system-5.

Sequence and Features