Index
Overview
In our project, we developed a detector to diagnose the major depressive disorder (MDD) by detecting microRNAs (miRNAs) miR-34a-5p, miR-221-3p and let-7d-3p, which were reported that their expressional changes in peripheral blood are as same as in cerebrospinal fluid (CSF) of patients. This detector uses a paper strip biosensor to detect miRNA based on toehold switch and cell free expression system, which facilitate the intuitive diagnosis for MDD at remote areas, resource limited environment or even at home for privacy protection.
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1. Construction and identification of positive controls for toehold switch plasmids
In order to make MDD diagnosis more intuitive and visible, we used some chromoproteins as the reporter proteins. They are amilCP (BBa_K2321010), mRFP (BBa_J04450) and amilGFP (BBa_K2321011) which exhibit blue/purple, red and yellow respectively. We constructed three new recombinant plasmids pET-28a-amilCP, pET-28a-mRFP and pET-28a-amilGFP to serve as positive controls, using the parts above.
These plasmids (pET-28a-amilCP, pET-28a-mRFP and pET-28a-amilGFP) were identified by restriction endonuclease digestion with EcoRⅠ and Hind Ⅲ, as well as PCR assay according to the flanking sequences of amilCP, mRFP and amilGFP, respectively. The Gene fragment lengths of amilCP, mRFP and amilGFP were about 701bp, 714bp and 732bp, respectively. Using the agarose electrophoresis, we found that their fragment lengths were consistent with the expected results (Fig.1, Fig.2 and Fig.3). These chromoproteins serve as reporter proteins for the construction and expression of toehold switch plasmids.
Fig.1 The identification of pET-28a-amilCP plasmid by restriction endonuclease digestion and PCR assay.
M: Marker, 1: Plasmid, 2: The plasmid digested by EcoRⅠ and Hind Ⅲ restriction endonuclease, 3: The amilCP gene amplified by PCR method.
Fig.2 The identification of pET-28a-mRFP plasmid by restriction endonuclease digestion and PCR assay.
M: Marker, 1: Plasmid, 2: The plasmid digested by EcoRⅠ and Hind Ⅲ restriction endonuclease, 3: The mRFP gene amplified by PCR method.
Fig.3 The identification of pET-28a-amilGFP plasmid by restriction endonuclease digestion and PCR assay.
M: Marker, 1: Plasmid, 2:The plasmid digested by EcoRⅠ and Hind Ⅲ restriction endonuclease, 3: The amilGFP gene amplified by PCR method.
The BL21 strains transfected with three plasmids (pET-28a-amilCP, pET-28a-mRFP and pET-28a-amilGFP), respectively, were cultured on the agar plates (Fig.4), showing the chromoproteins were expressed normally. These bacteria were also used as positive controls for subsequent toehold switch plasmids transfection assay.
Fig.4 Expression of chromoproteins in BL21 bacteria transfected with pET-28a-amilCP, pET-28a-mRFP and pET-28a-amilGFP plasmid, respectively.
1: Expression of amilCP gene, 2: Expression of mRFP gene, 3: Expression of amilGFP gene.
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2. Construction and identification of toehold switch plasmids
Based on the sequences of amilCP, mRFP and amilGFP, the complementary sequences of miR-34a-5p, miR-221-3p and let-7d-3p, and the construction rules of toehold switch, we designed three toehold switch plasmids to express amilCP, mRFP and amilGFP reporter genes, producing purple, red and yellow reporter proteins, respectively (Please refer to Design on our wiki for details). The toehold switches were synthesized and cloned into pET-28a expression vector through EcoR I-Hind III cloning Sites. They were named as pET-28a-toehold switch-amilCP, pET-28a-toehold switch-mRFP and pET-28a-toehold switch-amilGFP, which were designed to detect the amount of miR-34a-5p, miR-221-3p and let-7d-3p, respectively.
These toehold switch plasmids were identified by restriction endonucleases (EcoR I and Hind III) digestion assay. The inserted fragment lengths were 750, 762 and 780bp, respectively. Using agarose electrophoresis, we found that the inserted fragment lengths were consistent with the expected results, indicating that these toehold switch plasmids were constructed successfully (Fig.5).
Fig.5 Identification of pET-28a-toehold switch-amilCP, pET-28a-toehold switch-mRFP and pET-28a-toehold switch-amilGFP.
M: Marker, 1: pET-28a-toehold switch-amilCP plasmid digested byEcoR I and Hind Ⅲ restriction endonucleases, 2: pET-28a-toehold switch-amilCP plasmid, 3: pET-28a-toehold switch-mRFP plasmid digested by EcoRⅠ and Hind Ⅲ restriction endonucleases, 4: pET-28a-toehold switch-mRFP plasmid, 5: pET-28a-toehold switch-amilGFP plasmid digested by EcoRⅠ and Hind Ⅲ restriction endonucleases, 6: pET-28a-toehold switch-amilGFP plasmid.
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3. Expression of reporter proteins in BL21 strain with toehold switch plasmids and miRNAs transfection
The pET-28a-toehold switch-amilCP plasmid was transfected into BL21 strain (plate 2 in Fig.6). Since there is no miRNA 34a-5p in BL21 strain to trigger the expression of amilCP gene, it failed to express amilCP protein, or amilCP’s leakage expression was very low which cannot be observed with naked eyes, indicating that the toehold switch was effective. Next, the trigger miRNA (miR-34a-5p) was transfected into BL21 strain containing pET-28a-toehold switch-amilCP plasmid. We found that some clones successfully transfected with miR-34a-5p exhibited purple color, while those not successfully transfected with miRNA did not show the color (plate 3 in Fig.6). Plate 1 in Fig.6 served as a positive control (BL21 strain containing pET-28a-amilCP plasmid).
Fig.6 Expression of amilCP protein in BL21 strain.
1: Positive control of BL21 strain containing pET-28a-amilCP plasmid. 2: BL21 strain transfected only with pET-28a- toehold switch-amilCP plasmid, 3: BL21 strain transfected with pET-28a-toehold switch-amilCP plasmid and miR-34a-5p.
The pET-28a-toehold switch-mRFP plasmid was transfected into BL21 strain (plate 2 in Fig.7). Since there is no miRNA 221-3p in BL21 strain to trigger the expression of mRFP gene, it failed to express mRFP protein, or mRFP’s leakage expression was very low which cannot be observed with naked eyes, indicating that the toehold switch was effective. Next, the trigger miRNA (miR-221-3p) was transfected into BL21 strain containing pET-28a-toehold switch-mRFP plasmid. We found that some clones successfully transfected with miR-221-3p exhibited red color, while those not successfully transfected with miRNA did not show the color (plate 3 in Fig.7). Plate 1 in Fig.7 served as a positive control (BL21 strain containing pET-28a-mRFP plasmid).
Fig.7 Expression of mRFP protein in BL21 strain.
1: Positive control of BL21 strain containing pET-28a-mRFP plasmid. 2: BL21 strain transfected only with pET-28a- toehold switch-mRFP plasmid, 3: BL21 strain transfected with pET-28a-toehold switch-mRFP plasmid and miR-221-3p.
The pET-28a-toehold switch-amilGFP plasmid was transfected into BL21 strain (plate 2 in Fig.8). Since there is no miRNA let-7d-3p in BL21 strain to trigger the expression of amilGFP gene, it failed to express amilGFP protein, or amilGFP’s leakage expression was very low which cannot be observed with naked eyes, indicating that the toehold switch was effective. Next, the trigger miRNA (let-7d-3p) was transfected into BL21 strain containing pET-28a-toehold switch-amilGFP plasmid. We found that some clones successfully transfected with let-7d-3p exhibited yellow color, while those not successfully transfected with miRNA did not show the color (plate 3 in Fig.8). Plate 1 in Fig.8 served as a positive control (BL21 strain containing pET-28a-amilGFP plasmid).
Fig.8 Expression of amilGFP protein in BL21 strain.
1: Positive control of BL21 strain containing pET-28a-amilGFP plasmid. 2: BL21 strain transfected only with pET-28a- toehold switch-amilGFP plasmid, 3: BL21 strain transfected with pET-28a-toehold switch-amilGFP plasmid and let-7d-3p.
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4. Optimization of culture conditions of BL21 strains with toehold switch plasmids and miRNA transfection
After we got these BL21 strains transfected with toehold switch plasmids (pET-28a-toehold switch-amilCP, pET-28a-toehold switch-mRFP and pET-28a-toehold switch-amilGFP), we optimized BL21 strains culture conditions with trigger miRNAs (miR-34a-5p, miR-221-3p and let-7d-3p) for yielding more reporter protein production to increase the sensitivity of miRNA detection.
The BL21 strains containing toehold switch plasmids and certain miRNA were cultured under different conditions for optimization. Since reporter proteins have color, we can easily find the optimal conditions through the change of color. We selected different pH value, fermentation time, temperature and the concentration of transfected miRNA to optimize, respectively, and intuitively found the optimal conditions for obtaining the most yielding of reporter proteins by color changes (Fig.9, Fig.10, Fig.11 and Fig.12).
Fig.9 The effect of pH value of culture media on the expression of reporter proteins in BL21 strains with toehold switch plasmids transfected with their corresponding miRNAs.
(A) BL21 strain with pET-28a-toehold switch-amilCP transfected with miR-34a-5p, (B) BL21 strain with pET-28a-toehold switch-mRFP transfected with miR-221-3p, (C) BL21 strain with pET-28a-toehold switch-amilGFP transfected with let-7d-3p. 1: pH6, 2: pH6.4, 3: pH6.8, 4: pH7.0, 5: pH7.2, 6: pH8.0, 7: pH8.4.
Fig.10 The effect of temperature on the expression of reporter proteins in BL21 strains with toehold switch plasmids transfected with their corresponding miRNAs.
(A) BL21 strain with pET-28a-toehold switch-amilCP transfected with miR-34a-5p, (B) BL21 strain with pET-28a-toehold switch-mRFP transfected with miR-221-3p, (C) BL21 strain with pET-28a-toehold switch-amilGFP transfected with let-7d-3p. 1: 27℃, 2: 29℃, 3: 31℃, 4: 33℃, 5: 35℃, 6:37℃, 7: 39℃.
Fig.11 The effect of fermentation time on the expression of reporter proteins in BL21 strains with toehold switch plasmids transfected with their corresponding miRNAs.
(A) BL21 strain with pET-28a-toehold switch-amilCP transfected with miR-34a-5p, (B) BL21 strain with pET-28a-toehold switch-mRFP transfected with miR-221-3p, (C) BL21 strain with pET-28a-toehold switch-amilGFP transfected with let-7d-3p. 1: 8h, 2: 10h, 3: 12h, 4: 14h, 5: 16h, 6: 18h, 7: 20h.
Fig.12 The effect of miRNA concentration on the expression of reporter proteins in BL21 strains with toehold switch plasmids transfected with their corresponding miRNAs.
(A) BL21 strain with pET-28a-toehold switch-amilCP transfected with miR-34a-5p, (B) BL21 strain with pET-28a-toehold switch-mRFP transfected with miR-221-3p, (C) BL21 strain with pET-28a-toehold switch-amilGFP transfected with let-7d-3p. 1: 0.1uM, 2: 0.25uM, 3: 0.5uM, 4: 0.75uM, 5: 1uM, 6:1.5uM, 7: 2uM.
The optimization experiment results indicated that pH7.2, 37°C, fermentation 18h, and 1.5uM miRNA are the best culture conditions for higher reporter protein production in E. coli BL21 strains.
These experiments provided a foundation for the subsequent preparation of cell free system to improve the protein expression efficiency, increasing the sensitivity of detection.
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5. Preparation of cell free system and optimization of reaction conditions
After culturing BL21 strains transfected with toehold switch plasmids under the optimal culture conditions mentioned above, the cell extract was obtained using ultrasonication. The cell free expression system was prepared by mixing the cell extract with other components such as ATP, phosphoenolpyruvate (PEP), amino acid, etc. (please refer protocol section for details), and then added its corresponding trigger miRNA. In order to obtain sensitive and fast detection effects, the reaction conditions for expressing reporter proteins in cell free expression system were optimized under different temperature, rotation rate and reaction time. We took BL21 strain transfected with pET-28a-toehold switch-amilCP plasmid and its corresponding cell free expression system as an example to perform optimization experiments.
Fig.13 The optimization of temperature for the reporter protein expression in cell free system.
Fig.14 The optimization of rotation rate for the reporter protein expression in cell free system.
Fig.15 The optimization of reaction time for the reporter protein expression in cell free system.
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6. Preparation of paper strip based on the cell free system and the reporter gene expression on it
Filter paper is a ubiquitous, low-cost, easy to manufacture, store or transport biological analysis materials. After the filter paper was blocked with bovine serum albumin (BSA), washed and dried, a drop of the cell free reaction system according to the formula was added onto the filter paper, which was followed to put into the ultra-low temperature refrigerator and frozen dryer to form a paper strip sensor. When a 6μL water solution containing a certain miRNA was dropped onto the paper strip sensor containing its corresponding toehold switch plasmid, the protein expression was activated under the static conditions at 30°C for 1h.
Fig.16 Reporter gene expression triggered by different concentration of miRNA on the paper strip sensor based on the cell free reaction system.
(A) Expression of reporter gene amilCP triggered by different concentration of miR-34a-5p, (B) Expression of reporter gene mRFP triggered by different concentration of miR-221-3p, (C) Expression of reporter gene amilGFP triggered by different concentration of let-7d-3p.
The results showed that the concentration of target miR-34a-5p, miR-221-3p and let-7d-3p used to activate their corresponding toehold switch sensors were 10 ng/μL, 15 ng/μL and 15 ng/μL (Fig.16), respectively.
In order to increase the detection sensitivity, the reporter gene amilCP in the toehold switch plasmid was replaced with LacZ gene, expecting to amplify the reaction signal by the enzyme reaction that X-gal is cleaved to produce 5-bromo-4-chloro Indigo (blue color) catalyzed by β-galactosidase.
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7. Construction and identification of pET-28a-toehold switch-LacZ plasmid
Based on the flanking sequences of amilCP in pET-28a-toehold switch-amilCP plasmid, the amilCP gene was replaced with LacZ gene (3075bp) at EcoRⅠandHind Ⅲ cloning sites using PCR method, to construct the recombinant plasmid pET-28a-toehold switch-LacZ. For identification of this plasmid, the restriction endonuclease digestion and PCR assay were performed. The results showed that the fragment length of lacZ was consistent with the expected results, indicating that pET-28a-toehold switch-lacZ was constructed successfully (Fig.17)
Fig.17 Identification of pET-28a-toehold switch-LacZ plasmid.
M: Marker, 1: The plasmid of pET-28a-toehold switch-LacZ, 2: The pET-28a-toehold switch-LacZ plasmid was digested by EcoRⅠ and Hind Ⅲ restriction endonuclease, 3: The LacZ gene amplified by PCR method.
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8. Obtaining Kanamycin resistant BL21 strain with LacZ gene deletion by homologous recombination
For transfection with pET-28a-toehold switch-LacZ, we constructed BL21 mutant strain with LacZ gene deletion (BL21DLacZ). For amplification kanamycin gene to replace LacZ gene of BL21 genome, PCR primers were designed and the 5’ end of forward and reverse primers were added with the start and end sequences of LacZ gene, respectively. Using pET-28a vector as a template, the kanamycin gene (1.6Kb) was obtained by PCR amplification, which was shown in Fig.18.
Due to the design of primers, the amplified kanamycin gene was flanked with the start and end sequences of LacZ gene. Then the PCR product was transfected into BL21 strain in which homologous recombination happened between the PCR product and the LacZ gene of BL21 genome, obtaining BL21 mutant strain with LacZ gene deletion (BL21DLacZ). This mutant strain was screened out with kanamycin and confirmed by PCR results of LacZ and kanamycin genes (Fig.19), indicating that the Kanamycin resistant BL21 strain with LacZ gene deletion (BL21DLacZ) was obtained successfully.
Fig.18 The PCR result of Kanamycin gene using pET-28a vector as a template.
This PCR product of kanamycin gene was flanked with the start and end sequences of LacZ gene. M: Marker, 1: Kanamycin gene amplified by PCR method.
Fig.19 The PCR results of Kanamycin and LacZ genes in kanamycin resistant BL21DLacZ strain.
M: Marker, 1: Kanamycin gene amplified by PCR method. 2. No band of LacZ gene amplified by PCR method.
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9. Preparation of paper strip biosensor with enzyme reaction system for β-galactosidase
pET-28a-toehold switch-LacZ plasmid was transfected into BL21ΔLacZ strain. Under the optimal conditions mentioned above, cell extract was obtained by the strain collection and ultrasonication. The cell free expression system was prepared by mixing the cell extract with other components such as ATP, PEP, amino acid, etc. (please refer to the Experiment section for details). After the filter paper was blocked with bovine serum albumin (BSA), washed and dried, a drop of the cell free reaction system mentioned above fell onto the filter paper strip which then was put into the ultra-low temperature refrigerator and frozen dryer to form a paper strip biosensor.
In order to obtain sensitive and fast detection effects, the reaction conditions under which X-gal is cleaved to produce 5-bromo-4-chloro Indigo (blue color) catalyzed by β-galactosidase in the cell free expression system were optimized under different temperature, reaction time and miRNA concentration.
Temperature is the primary factor affecting enzyme reaction in vitro. The optimization results showed that the temperatures of cell free expression system and enzyme reaction ranged from 25 °C to 42 °C, but the best temperature was 30°C, as shown in Fig.20. When the reaction lasted for 1h, the reaction was almost over, so 1h was chosen as the best reaction time (Fig.21). With the decrease of miRNA concentration, the color of reaction solution gradually changed from positive blue to negative colorless. For miR-34a-5p target sensor, the lowest limit of visible color development is 500fM (Fig.22).
Fig.20 The optimization of temperature for reactions catalyzed by β-galactosidase in cell free system.
(A): OD570 value absorbed by the blue product, (B): Photograph of paper strip biosensor reaction in cell free system.
Fig.21 The optimization of reaction time for β-galactosidase catalyzing reaction in cell free system.
(A): OD570 value absorbed by the blue product, (B): Photograph of paper strip biosensor reaction in cell free system.
Fig.22 The optimization of miR-34a-5p concentration for β-galactosidase catalyzing reaction after 1h.
(A): OD570 value absorbed by the blue product, (B): Photograph of paper strip biosensor reaction in cell free system.
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Conclusion
We established a paper strip biosensor used for a detector to diagnose MDD by detecting miR-34a-5p, miR-221-3p and let-7d-3p. When using the chromoproteins as reporters, the lowest detecting concentration of target miRNAs is 10 ng/μL, 15 ng/μL and 15 ng/μL, respectively. When using β-galactosidase as a reporter, the lowest detecting concentration of target miR-34a-5p is 500fM, which increased more sensitivity.
This simple and convenient detector with filter paper strip biosensor is a powerful supplement to the current diagnosis of MDD which depends on clinical symptoms and self-assessment scale. This detector is very beneficial to the objective evaluation of MDD diagnosis. And people can use it to diagnose for himself at home for privacy protection due to its simplicity and portability.
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Future plan and considerations
1.This detector with filter paper strip biosensor would be used for diagnosis of MDD using serum or blood as samples, so these samples should be applied to test its practicability in the future experiment.
2.To increase the sensitivity of the detection, isothermal amplification of nucleic acid should be explored in the future, which can amplify the amount of miRNA in the samples such as serum or blood of the patients.
3.Most data in our project were rough due to the time limitation and our ability. Some experiments need to be replicated and modified for obtaining reliable results.
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