Team:OUC-China/Demonstrate
Demonstrate
To help you enjoy our result in a more convenient and smooth way, here are some key concepts in our discussion.
1) miniToe system
2) miniToe family system (The miniToe family refers to the whole second system below)
3) miniToe polycistron system (The miniToe polycistron refers to the whole third system below)
4) miniToe based motility detection system
The first system (miniToe system) is the basics of other three systems. As description we introduced above, on the level of DNA, we design a miniToe part and construct it to circuits. On the level of RNA, we focus on miniToe structure. In the miniToe family system, we focus on Csy4 hairpin which is a part of miniToe part. In fact, the Csy4 hairpin is the miniToe target region. A cis-repressive RNA (crRNA) served as a translation suppressor by pairing with RBS is critical part of miniToe structure.
Also, the miniToe system is a kind of translation activator. In miniToe family, the capabilities of different Csy4 mutants reflect their activation efficiency to turn on translation. So the fluorescence intensity reflect activation efficiency which is a comprehensive ability including the capabilities of recognition and cleavage.
1. The first system: miniToe
1.1 New method: miniToe
Based on function of Csy4, we design a new cis-regulatory RNA element named miniToe which can be recognized by Csy4 [1]. The whole system works as a translational activator including three modular parts:
1. A cis-repressive RNA (crRNA) served as a translational suppressor by pairing with RBS as the critical part of miniToe structure.
2. A Csy4 site as a linker between cis-repressive RNA and RBS, which can be specifically cleaved upon Csy4 function.
3. A CRISPR endoribonuclease Csy4.
Fig.1 The composition of miniToe system.
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.2 The two plasmids of miniToe system.
Without Csy4, the crRNA 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 downstream gene can be regulated.
Fig.3 The mechanism of miniToe system.
1.2 Proof of function
There are two problems that need to be proved about miniToe system.
Firstly, 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 [2][3].
Fig.4 The structure prediction of full-length transcript of this circuit as well as miniToe structure. The miniToe structure is on the left of picture and the full-length transcript of this circuit is on the right of picture. The red frame indicates the places of miniToe structure in the full-length transcript of this circuit.
As the result showed in Fig.5, a control group (the green line) is relatively stable during the whole process comparing with other another control group (IPTG=0) and test group(IPTG=0.125mM). 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.
Fig.5 The fluorescence intensity of sfGFP by microplate reader during the entire cultivation period. There are three groups which means three different strains tested in the chart. The yellow line is a test group with IPTG (0.125mM). The blue line is a control group without IPTG (0mM). The green line is a control group with only one plasmid (pReporter).
The second problem that needs to be proved is whether miniToe system can work successfully and regulate the downstream genes expression as a switch. Obviously, in the Fig.5, there is a rise in expression of sfGFP between two lines in the whole process. The yellow line is the test group with 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 the fact that the expression of target gene sfGFP increases indeed due to cleavage of Csy4 site, thus exposing the RBS and restarting translation. It means miniToe system can work successfully.
We also test miniToe system by flow cytometric. In Fig.6, it's easy to distinguish the two groups (blue & white).The intensity of sfGFP of the test group (+IPTG) increases obviously compared with the control group (IPTG=0). The result shows the same conclusions mentioned before.
Fig.6 Flow cytometric measurement the intensity 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 intensity. 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).
Fig.7 The results from other four teams which proved our conclusions. Error bars represent standard deviation of four biological replicates.
1.3 The characteristics of miniToe system
1. The Csy4 protein and miniToe structure have high binding affinity [4]. It shows that the miniToe system may control the state of expression in a digital-like way (ON/OFF). When the switch is at OFF state, the downstream gene expression is tightly blocked, the reaction grows very slowly in the beginning but accelerating rapidly once the Csy4 protein truncates the cis-repressive RNA element.
2. Compared to the small RNA based on riboswitch [5], the insertion of hairpin provides Csy4 with a recognition and cleavage site so that the Csy4 may enhance the steric hindrance effect between cis-repressive RNA and RBS when we need to release the cis-repressive RNA for opening the downstream gene expression, which could promote translation activation.
3. Compared to the toehold switch [6], miniToe does not need to redesign cis-repressive RNA case by case because the cis-repressive RNA is not paired with protein coding region.
2. The second system: miniToe family
2.1 The principles of designing mutants
For Csy4 mutants
1. Some key sites in the Csy4 are really crucial for keeping the stability of the structure and maintaining the functions of recognition and cleavage. Mutations on those sites may have a serious influence on our system. By point mutation, we hoped to get a library of mutants, which could provide several Csy4 mutant candidates with recognition and cleavage rates showing as a "ladder".
For hairpin mutants
2. We need to avoid breaking the recognition site and keep the function of cleavage relatively stable. If we break the key site G20, it may lead to the damage of cleavage function.
3. The stability of secondary structure is vital, so we need to focus on each hairpin's Gibbs free energy during designing.
4. The aim is to obtain different hairpins which have various activity for Csy4 recognition and RNA cleavage.
With the help of our model, 4 Csy4 mutants and 5 hairpin mutants are selected. We tested each mutant and got positive data supporting model prediction. Then we set up a function verification experiment with 5*6 combinations of Csy4 and hairpin including wild types. By testing all of them, 10 combinations work successfully as expectation. So, our second system, miniToe family, has 10 combinations which were well designed and selected.
2.2 Proof of functions
First, we tested the capabilities of five Csy4 mutants:
1. The result by Microscope
2. The result by flow cytometer
3. The result by microplate reader
Second, six different hairpin mutants were tested by microplate reader.
Finally, all the 30 groups' intensities of fluorescence is tested. We ranked them by the heat map and then selected the groups from different expression levels. As you can see, in the heat map, the expression levels of some groups are almost the same. So we just give up some combinations and then select the groups we really need to be the members of miniToe family. The user-friendly system meets the flexible needs in study about regulating different levels of expression. The final 10 members of miniToe family are shown below.
We designed three kinds experiments to test the capabilities of five Csy4 mutants by putting them into miniToe system. So the recombination strains for test both have same pReporter but different Csy4 mutants plasmids in the following. The recombination strains to test the functions of Csy4 are strain-Csy4 (pCsy4&pReporter), strain-Csy4-Q104A (pCsy4-Q104A&pReporter), strain-Csy4-Y176F (pCsy4-Y176F&pReporter), strain-Csy4-F155A (pCsy4-F155A&pReporter), strain-Csy4-H29A (pCsy4-H29A&pReporter). At the same time, we have a control strain named strain-miniToe-only which only has pReporter.
2.2.1 Proof of functions: Csy4 mutants
The qualitative experiment by Microscope. We can observe visible distinctions in these images. The fluorescence intensities decrease one by one from top to bottom which means the Csy4s' capabilities of cleavage decrease one by one. Their order goes from strong to weak is Csy4-WT, Csy4-Q104A, Csy4-Y176F, Csy4-F155A and Csy4-H29A.
1. The expression of sfGFP by strain-Csy4-WT.
2. The expression of sfGFP by strain-Csy4-Q104A.
3. The expression of sfGFP by strain-Csy4-Y176F.
4. The expression of sfGFP by strain-Csy4-F155A.
5. The expression of sfGFP by strain-Csy4-H29A.
Fig.8 The fluorescence images by fluorescent microscope. From top to bottom, the images shows the expression of sfGFP by strain-Csy4, strain-Csy4-Q104A, strain-Csy4-Y176F, strain-Csy4-F155A and strain-Csy4-H29A in sequence. 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.
Fig.9 The intensities of sfGFP of Csy4 mutants by flow cytometer. Histograms show distribution of fluorescence in samples with strain-Csy4 (Black), strain-Csy4-Q104A (Orange), strain-Csy4-Y176F (Red), strain-Csy4-F155A (Blue), strain-Csy4-H29A (Green). Crosscolumn number shows fold increase of sfGFP fluorescence.
Fig.10 The Gate Mean of flow cytometer. Histograms show the relative expression of sfGFP. The five test groups present different fluorescence intensities from high to low, which proves that they have different capabilities.
Fig.11 The comparision about model and result by microplate reader. The intensities of sfGFP by microplate reader on the left when the model is on the right.
By all the experiments mentioned before, we proved that Csy4 mutants work as expectations successfully. The results are listed in the order: Csy4-WT>Csy4-Q104A>Csy4-Y176F>Csy4-F155A>Csy4-H29A. And the original sequences of Csy4 part has been submitted by other iGEM teams before, so this year we improved their work by enlarging Csy4 to a Csy4 family.
2.2.2 Proof of functions: Hairpin mutants
We also redesigned 5 hairpin mutants and tested them by flow cytometry and ranked them by their capabilities. The results are listed in the order: miniToe-WT>miniToe-5>miniToe-1>miniToe-4>miniToe-2>miniToe-3.
Fig.12 The intensities of sfGFP about hairpin mutants by flow cytometer. Histograms show distribution of fluorescence in samples with strain-miniToe (Black), strain-miniToe-5 (Red), strain-miniToe-1 (Green), strain-miniToe-4 (Blue), strain-miniToe-2 (Cyan), strain-miniToe-3 (Yellow). Crosscolumn number shows fold increase of sfGFP fluorescence.
Fig.13 The Gate Mean of flow cytometer. Histograms show the relative expression of sfGFP. The six test groups present different fluorescence intensities from high to low, which proves that they have different capabilities.
2.2.3 Proof of functions: MiniToe family
And the whole system is tested by flow cytometry. All the 30 groups' intensities of fluorescence are shown in Fig.14. We ranked them by the heat map and then selected the groups from different expression levels. As you can see, in the heat map, the expression levels of some groups are almost the same. So we just gave up some combinations and then selected the groups we really need to be the members of miniToe family. The final 10 members of miniToe family are shown in the Fig.15. The user-friendly system meets the flexible needs in study which can meet user's need about different levels of expression.
Fig.14 The heat map generated from flow cytometry data reflecting intensities of fluorescence by 30 combinations.
Fig.15 The members of miniToe family.
3. The third system: miniToe polycistron
3.1 The design of miniToe ploycistron
Many applications of synthetic biology need the balanced expressions of multiple genes. For the sake of tuning the expression of genes in polycistron, we planed to realize a tightly regulation by the miniToe structure. Our aim is achieving different proportions of output by miniToe in polycistrons compared with normal polycistrons.
Fig.16 The mechanisms of miniToe polycistron.
By inserting miniToe hairpins between intergenetic regions, it will tune the translation level of corresponding proteins.
1) First, sfGFP and mCherry is used as a test system in bi-cistron circuit.
2) Then we selected some miniToe parts and inserted them between, before and behind sfGFP and mCherry. For example, for bi-cistron, then three miniToe parts will be inserted. For three genes in polycistron, then four miniToe parts will be inserted, and so on.
Two kinds of groups are set. One is the bicistron circuit without miniToe parts. The other is the test group which have miniToe parts. This year, we designed two kinds of miniToe polycistrons, miniToe polycistron-A and miniToe polycistron-B. In the future, we will test more polycistron based on miniToe family.
Fig.17 The two groups in experiment. Group A is the control group without miniToe system. Group B is the test group with miniToe system.
3.2 Proof of functions
We tested our miniToe polycistron by microplate reader. The sfGFP were measured at excitation/emission wavelengths of 485nm/520nm. The mCherry were measured at excitation/emission wavelengths of 587nm/610nm.
Fig.18 The rate of fluorescence intensities by sfGFP/mCherry.
4. THE FOURTH SYSTEM: MINITOE BASED MOTILITY DETECTION SYSTEM
4.1 The purpose of designing the experiment
As a translation regulation tool, miniToe can also be used in application scenario of molecular mechanism study. Sometimes scientists may puzzle about the functions of certain gene or protein without in-depth study. One general method to study them is to "knock-out" or "knock-in". In this way, organisms show some phenotypic changes related to particular gene. However, if we want to know better about the functions of the gene, we may need more tools to realize different levels of gene expression.
By using our system, the motility of E. coli can be regulated. As we all know, MotA provides a channel for the proton gradient required for generation of torque [8]. ΔmotA strains (the motA -deletion strain) can build flagella but are non-motile because they are unable to generate the torque required for flagellar rotation.
We did a lot of works to test our minToe system by applying it to the detection of E. coli motility. We constructed circuit by putting the motA behind miniToe part. So the target gene motA can be regulated by our miniToe system.
Fig.19 The process of motility detection system
4.2 Proof of functions
Five groups are set, a test group and four control groups. And the results shown below proved that our system can work as expectation.
Fig.20 The control groups A and B including positive group and negative group. Plates were inoculated with E. coliRP437 (A1, A2, A3) that have motility and they can move arbitrarily in the plates. The plates on right are ΔmotA strains(the motA-deletion strain) (B1, B2, B3), E. coli RP6666, which have no motility so the strains stay on the center. We have three biological replicates in this experiment.
Fig.21 The test group C. The plates were inoculated with Csy4-ΔmotA (the motA-deletion strain with Csy4 but no miniToe structure).Without the gene motA, the E. coli cannot move. And the Csy4 have no big influence on strain compared with the ΔmotA strain. The little round of papers indicates the places of inducer IPTG (Isopropyl-β-d-thiogalactoside). We have three biological replicates in the experiment.
Fig.22 The test group D. The plates were inoculated with miniToe-motA (the motA-deletion strain with miniToe structure but no Csy4. The circuit is on the control of miniToe and its downstream gene motA can be regulated without Csy4. So the expression of downstream gene motA keep closing. We have three biological replicates in the experiment.
Fig.23 The test group E. The strain we cultured in plates is miniToe-motA with Csy4. The strain have the whole miniToe system which means motA can be regulated by miniToe part. In the picture, the E. coli moved everywhere in the plates, proving that with the regulation of miniToe and Csy4, the downstream gene motA comes into play. The E. coli can move everywhere in the plate. We have three biological replicates in the experiment.
Fig.24 The migration dimensions. The ratio of migration area /whole plate. This chart is made by numerical integration.
As we can see, test group strains can move everywhere in the plate and the control groups strains cannot move. The test group works as expectation compared to the control groups. But there is no time for us to test more miniToe mutants and Csy4 mutants in miniToe family. We want to realize the function of regulation by using different miniToe family members in the future. So we still have a lot of work to do.
5. Improvement based on 2016 OUC-China
The 2016 OUC-China had a method to regulate the expression of polycistron.
They concentrated on stem-loops inserted into the intergenic regions. When transcribed as one polycistron, digested and separated into several independent fragments, cistrons with 3' end stem-loops will get different stability. They designed a lot of different stem-loop. And they measured and standardized a series of native and designed stem-loops, transforming into a toolkit for better uses.
Click here to see 2016 OUC-China.
Fig.25 The three stem-loops designed by 2016 OUC-China
The new system named miniToe polycistron is an important improvement inspired by 2016 OUC-China. The idea using stem-loop to regulate the gene expression is creative and inspired us to do more. We found miniToe part is also a good stem-loop that can be inserted to polycistron. We inserted three miniToe parts to circuit when we need to tune the expression of two genes. It means that for each target gene we have two miniToe hairpins. The one is downstream of the gene, and the other is upstream of the gene. We made an enhanced version based on previous project.
1.The 2016 OUC-China only put the hairpins between two genes, and we put the hairpins downstream and upstream for each gene. Without Csy4, the existence of miniToe structure in polycistron will inhibit the gene expression when we don’t want to open the switch. The cis-repressive RNA in miniToe has complementary sequence of adjacent RNA region (RBS) to prevent the binding of ribosomes.
2. After cleavage, in the 5' end, the capabilities of cleavage by RNase E is much lower because there is no pyrophosphate in the 5' end. Qi's work has proved that OH-mRNAs exhibit higher gene expression than 5' PPP-mRNAs [7].
3. Also, the RNA secondary structure of miniToe with Csy4 binding keeping at 3’ UTR after Csy4 cleavage is a protection mechanism to prevent RNA degradation. Because more energy is needed which partly provided by ATP to degrade the RNA/Csy4 complexes in the 3' end, the degradation rate of RNA is much lower. And this function is based on the high stability and affinity [7] between Csy4 and miniToe structure.
4. The function of each miniToe structure has specific recognition and cleavage rates, which will make it possible to regulate the gene flexibly.
ACHIEVEMENTS
★Our engineered system worked under realistic conditions, which is proved by our different forms of experimental datas, including the qualitative experiment by microscope, flow cytometric measurements and microplate reader. Furthermore, we applied our system into motility detection system and it worked well. Other four iGEM teams also helped us test our system successfully in their lab.★We put safety in the first place all the time, ensuring the safety of our project, the safety of our lab work and the safety of biobrick shipment. Go to see Safety page!
★Our miniToe polycistron system derives from 2016 OUC-China. Based on their project, we put the hairpins downstream and upstream for each gene rather than only put the hairpins between two genes. In our system, the degradation rate of RNA hairpin is much lower than theirs, and the gene regulation is more flexible, which improved a lot compared to their project.
★We have designed four systems this year. We measured functions of these four systems in detail and describe it clearly.
Go to see design page!
Go to see result page!
REFERENCE
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[3].M. Zuker. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31 (13), 3406-3415, 2003.
[4].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.
[5].Mandal M, Breaker R R. Gene regulation by riboswitches.[J]. Nature Reviews Molecular Cell Biology, 2004, 5(6):451-63.
[6].Green A, Silver P, Collins J, et al. Toehold switches: de-novo-designed regulators of gene expression.[J]. Cell, 2014, 159(4):925-939.
[7].Qi L, Haurwitz R E, Shao W, et al. RNA processing enables predictable programming of gene expression[J]. Nature Biotechnology, 2012, 30(10):1002.
[8].Ravichandar J D, Bower A G, Julius A A, et al. Transcriptional control of motility enables directional movement ofEscherichia coliin a signal gradient[J]. Scientific Reports, 2017, 7(1).
