Overview
In our project, we developed a detector to perform the early warning of atherosclerosis (AS) by detecting trimethylamine N-oxide (TMAO) and microRNAs (miR-17-5p and miR-146a-5p), all of which were reported high concentration in the urine of patients. This detector uses a filter paper biosensor to detect TMAO and miRNAs based on TMAO receptor system and toehold switch, respectively, in cell free expression system. It facilitates the early warning of AS at home, timely treatment, and avoiding cardiovascular and cerebrovascular diseases (CCVD) caused by AS which is the most risk factor for elderly people health.
1. TMAO testing system
1.1 Construction and identification of expression vectors containing TorR, TorT and TorS genes
In order to make early warning of AS more intuitive and visible, we used beta-galactosidase (lacZ) as the reporter protein. We constructed two recombined plasmids: one of them is pET-28a-TorR-lacZ containing TorR gene with transcriptional terminator sequence followed by TorCAD promoter and lacZ gene, which means TorR and lacZ genes are transcribed by different promoters; another one is pET-22b-TorT-TorS containing TorT without transcriptional terminator and TorS gene, which means the two genes are regulated by the same promoter.
The TorR gene with transcriptional terminator sequence, TorCAD promoter and lacZ gene flanked with BamH I and Hind III digestion sites were synthesized, and then inserted into pET-28a plasmid (Kan+), constructing pET-28a-TorR-lacZ expression vector. Similarly, TorT without transcriptional terminator and TorS gene flanked with BamH I and Hind III digestion sites were also synthesized and inserted into pET-22b plasmid (Amp+), constructing pET-22b-TorT-TorS expression vector.
The two recombinant plasmids (pET-28a-TorR-lacZ and pET-22b-TorT-TorS) were identified by BamH I and Hind III digestion, as well as PCR assay according to the flanking sequences of TorR-lacZ and TorT-TorS, respectively. The gene fragment lengths of TorR-lacZ and TorT-TorS were about 3855 bp and 3744 bp, respectively. Using the agarose electrophoresis, we found that their fragment lengths were consistent with the expected results (Fig.1 and Fig.2). These two recombinant plasmids were used to detect TMAO by co-transformation to express lacZ.
Fig.1 Identification of pET-28a-TorR-lacZ plasmid.
M: Marker; 1: The plasmid of pET-28a-TorR-lacZ; 2: The pET-28a-TorR-lacZ plasmid was digested by BamH I and Hind Ⅲ restriction endonuclease; 3: The TorR-lacZ gene amplified with PCR method.
Fig.2 Identification of pET-28a-TorT-TorS plasmid.
M: Marker; 1: The plasmid of pET-28a-TorT-TorS; 2: The pET-28a-TorT-TorS plasmid was digested by BamH I and Hind Ⅲ restriction endonuclease; 3: The TorT-TorS gene amplified with PCR method.
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1.2 Expression and purification of TorR, TorT and TorS genes in BL21 (DE3) strain
The two recombinant plasmids pET-28a-TorR-lacZ and pET-22b-TorT-TorS were transformed into BL21 strain respectively, then cultured with fresh LB medium added Kanamycin and Ampicillin respectively. Soluble cytoplasmic proteins were extracted after isopropyl β-D-thiogalactoside (IPTG) induction 10 h. The recombinant proteins TorR, TorT and TorS were purified for identification using 6x His tag. Without TMAO, LacZ protein was not expressed since TorCAD promotor of LacZ can not be activated by TMAO. The results of expression and purification are showed in Fig.3 and Fig.4.
Fig.3 The SDS-PAGE result shows expression and purification of TorR protein.
M: Marker; 1: All supernatant proteins containing TorR induced by IPTG; 2: Supernatant proteins not bound to magnetic beads in purification process; 3: Proteins in wash buffer; 4: Purified TorR (27 kDa) protein in elution buffer.
Fig.4 The SDS-PAGE result shows expression and purification of TorT and TorS proteins.
M: Marker; 1: All supernatant proteins containing TorT and TorS induced by IPTG; 2: Supernatant proteins not bound to magnetic beads in purification process; 3: Proteins in wash buffer; 4: Purified TorT (40 kDa) and TorS (110 kDa) proteins in elution buffer.
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1.3 Construction and identification of Kanamycin resistant BL21 strain with LacZ gene deletion (BL21DLacZ)
To express the recombinant LacZ gene, we constructed BL21 (DE3) mutant strain with LacZ gene deletion (BL21DLacZ). For amplification kanamycin resistant gene to replace LacZ gene of BL21 genome, PCR primers were designed to be added with the start and end sequences of LacZ gene at the 5’-end of forward and reverse primers, respectively. Using pET-28a vector as a template, the kanamycin resistant gene (1.6Kb) was amplified with PCR method, which was shown in Fig.5. Due to the design of primers, the amplified kanamycin resistant gene was flanked with the start and end sequences of LacZ gene. Then the PCR product was transformed 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 using kanamycin and confirmed by PCR results of LacZ and kanamycin resistant genes (Fig.6), indicating that the Kanamycin resistant BL21 strain with LacZ gene deletion (BL21DLacZ) was constructed successfully.
Fig.5 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.
Fig.6 The PCR results of Kanamycin and LacZ genes in kanamycin resistant BL21DLacZ strain.
M: Marker； 1: Kanamycin gene. 2. No band of LacZ gene amplified with PCR method.
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1.4 Induced expression of LacZ triggered by TMAO in BL21DLacZ strain co-transformed with recombinant plasmids pET-28a-TorR-lacZ and pET-22b-TorT-TorS
The two plasmids pET-28a-TorR-lacZ and pET-22b-TorT-TorS were co-transformed into BL21DLacZ strain. Since there is no TMAO in BL21DLacZ culture medium to trigger the TorCAD promoter, it failed to express LacZ. Even at the presence of IPTG and X-gal, no blue clone was observed (Fig.7a). When TMAO was added into the agar medium, BL21DLacZ strain co-transformed with pET-28a-TorR-lacZ and pET-22b-TorT-TorS plasmids were observed blue clones at the presence of IPTG and X-gal (Fig.7b), indicating that this system can detect TMAO through the color change of clones.
Fig.7 The induced expression of LacZ in BL21DLacZ strain co-transformed with pET-28a-TorR-lacZ and pET-22b-TorT-TorS plasmids.
(a) Without TMAO trigger, LacZ was not induced expression, and no blue clone was observed at the presence of IPTG and X-gal. (b) With TMAO trigger, LacZ was induced expression, and many blue clones were observed at the presence of IPTG and X-gal.
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1.5 Optimization of reactional conditions catalyzed by β-galactosidase in cell free system to increase the sensitivity of TMAO detection
After co-transformation of pET-28a-TorR-lacZ and pET-22b-TorT-TorS plasmids into BL21DLacZ strain, it was cultured about 20 h with the induction of IPTG, then centrifuged and lysed with lysis buffer. The supernatant (cell free system) was collected after centrifugation again. Using the supernatant, we optimized the reaction conditions catalyzed by reporter protein (β-galactosidase) at the presence of TMAO and X-gal to increase the sensitivity of TMAO detection.
Since the reaction catalyzed by β-galactosidase has color change of blue, we can easily find the optimal conditions through the color change. We selected 4 different conditions including reaction temperature, time, concentration of X-gal and TMAO for optimization. The results were showed intuitively as follows (Fig.8), and performed quantitative analysis after 3 repetitions (Fig.9).
Fig.8 The optimization of reactional conditions catalized by β-galactosidase in cell free system to increase the sensitivity of TMAO detection.
(a) Reaction temperature: 1. 25 °C; 2. 27 °C; 3. 29 °C; 4. 31 °C; 5. 33 °C; 6. 35 °C; 7. 37 °C; 8. 39 °C. (b) Reaction time: 1. 5 min; 2. 10 min; 3. 15 min; 4. 20 min; 5. 30 min; 6. 40min; 7. 50 min; 8. 60 min. (c) Concentration of X-gal: 1. 12 µg/mL; 2. 16 µg/mL; 3. 24 µg/mL; 4. 30 µg/mL; 5. 40 µg/mL; 6. 60 µg/mL; 7. 120 µg/mL; 8. 240 µg/mL. (d) Detectable concentration of TMAO: 1. 20 µM; 2. 40 µM; 3. 60 µM; 4. 80 µM; 5. 100 µM; 6. 200 µM; 7. 400 µM; 8. 800 µM.
Fig.9 The quantitative analysis of conditional optimization.
(a). Optimization of reactional temperature; (b) Optimization of reaction time;
(c) Optimization of X-gal concentration; (d) Detectable concentration of TMAO.
The optimization experiment results showed that the best reaction conditions are 37 °C, 40 min and 40 µg/mL X-gal. The lowest visible detecting concentration of TMAO is 40 µM. These results provided a foundation for the subsequent experiment performed with cell free system on filter paper strip biosensor, which will be used in the detector to test TMAO concentration.
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2. MicroRNA testing system
2.1 Construction and identification of pET-28a miRNA toehold switch LacZ plasmids
To enhance the specificity of screening AS, we also constructed pET-28a miRNA toehold switch LacZ plasmids to detect microRNAs related to AS. According to the construction rules of toehold switch, two toehold switch plasmids pET-28a-miR-17-5p-LacZ and pET-28a-miR-146a-5p-LacZ were constructed for detecting miR-17-5p and miR-146a-5p, respectively.
For identification of these two plasmids, the restriction endonuclease (BamH I and Hind III) digestion and PCR assay were performed. The inserted fragment lengths were 3124 bp and 3123 bp, respectively. Using agarose electrophoresis, the results showed that the inserted fragment lengths were consistent with the expected results, indicating that the two toehold switch plasmids were constructed successfully (Fig.10 and Fig.11).
Fig.10 Identification of pET-28a-miR-17-5p toehold switch-lacZ plasmid.
M: Marker; 1: The plasmid of pET-28a-miR-17-5p toehold switch-lacZ; 2: The pET-28a-miR-17-5p toehold switch-lacZ plasmid was digested by BamH I and Hind III restriction endonuclease; 3: The inserted fragment containing LacZ gene amplified with PCR method.
Fig.11 Identification of pET-28a-miR-146a-5p toehold switch-LacZ plasmid.
M: Marker; 1: The plasmid of pET-28a-miR-146a-5p toehold switch-LacZ; 2: The pET-28a-miR-146a-5p toehold switch-LacZ plasmid was digested by BamH Ⅰ and Hind Ⅲ restriction endonuclease; 3: The inserted fragment containing LacZ gene amplified with PCR method.
2.2 Induced expression of LacZ by microRNAs in BL21DLacZ strain transformed with toehold switch plasmids
pET-28a-miR-17-5p toehold switch-lacZ and pET-28a-miR-146a-5p toehold switch-LacZ plasmids were transformed into BL21DLacZ strain respectively. Without miR-17-5p or miR-146a-5p to trigger the expression of LacZ gene, no β-galactosidase catalyzed X-gal to make clone blue (Fig.12a and Fig.13a). When the trigger miRNAs (miR-17-5p or miR-146a-5p) were transformed into BL21DLacZ strain containing pET-28a-miR-17-5p toehold switch-lacZ or pET-28a-miR-146a-5p toehold switch-LacZ, the LacZ expression was switched on to produce β-galactosidase, making some clones show blue (Fig.12b and Fig.13b), indicating that the construction of toehold switches were successful. They can be used for detection of miR-17-5p and miR-146a-5p. Both Fig.12c and Fig.13c served as positive controls.
Fig.12 Induced expression of LacZ gene by miR-17-5p in BL21DLacZ strain transformed with pET-28a-miR-17-5p toehold switch-lacZ plasmid.
(a) Without miR-17-5p, BL21DLacZ strain transformed with pET-28a-miR-17-5p toehold switch-LacZ plasmid can not produce β-galactosidase to make clones blue (showing white clones). (b) With trigger miR-17-5p, BL21DLacZ strain transformed with pET-28a-miR-17-5p toehold switch-LacZ plasmid expressed β-galactosidase, making some clones blue (some white clones indicated that they were not transformed successfully with miR-17-5p). (c) Positive control of BL21(DE3) strain at the presence of X-gal.
Fig.13 Induced expression of LacZ gene by miR-146a-5p in BL21DLacZ strain transformed with pET-28a-miR-146a-5p toehold switch-LacZ plasmid.
(a) Without miR-146a-5p, BL21DLacZ strain transformed with ET-28a-miR-146a-5p toehold switch-LacZ plasmid can not express β-galactosidase to make clones blue (showing white clones). (b) With trigger miR-146a-5p, BL21DLacZ strain transformed with pET-28a-miR-146a-5p toehold switch-LacZ plasmid expressed β-galactosidase, making some clones blue (some white clones indicated that they were not transformed successfully with miR-146a-5p). (c) Positive control of BL21(DE3) strain at the presence of X-gal.
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2.3 Optimization of reactional conditions catalyzed by β-galactosidase in cell free system to increase the sensitivity of microRNA detection
After transformation of pET-28a-miR-17-5p toehold switch-LacZ or pET-28a-miR-146a-5p toehold switch-LacZ plasmid into BL21DLacZ strain, they were cultured 16-20 h with the induction of IPTG (LacZ gene was transcribed but not translated), then centrifuged and lysed with lysis buffer. The supernatant (cell free system) was collected after centrifugation again. Using the supernatant, we optimized the reaction conditions catalyzed by reporter protein (β-galactosidase) at the presence of corresponding microRNA and X-gal to increase the sensitivity of microRNA detection.
Since the reaction catalyzed by β-galactosidase has color change of blue, we can easily find the optimal conditions through the color change. We selected 4 different conditions including reaction temperature, time, concentration of X-gal and certain microRNA for optimization. The results were showed intuitively in Fig.14 and Fig.16, which were performed quantitative analysis after 3 repetitions in Fig.15 and Fig.17, respectively.
Fig.14 The optimization of reactional conditions catalyzed by β-galactosidase in cell free system to increase the sensitivity of miR-17-5p detection.
(a) Reaction temperature: 1. 27 °C; 2. 29 °C; 3. 31 °C; 4. 33 °C; 5. 35 °C; 6. 37 °C; 7. 39 °C; 8. 41 °C. (b) Reaction time: 1. 2 min; 2. 5 min; 3. 10 min; 4. 15 min; 5. 20 min; 6. 25min; 7. 30 min; 8. 35 min. (c) Concentration of X-gal: 1. 12 µg/mL; 2. 16 µg/mL; 3. 24 µg/mL; 4. 30 µg/mL; 5. 40 µg/mL; 6. 60 µg/mL; 7. 120 µg/mL; 8. 240 µg/mL. (d) Detectable concentration of miR-17-5p: 1. 50 fM; 2. 500 fM; 3. 5 pM; 4. 50 pM; 5. 500 pM; 6. 5 nM; 7. 50 nM; 8. 100 nM..
Fig.15 The quantitative analysis of conditional optimization for detection of miR-17-5p using cell free system.
(a) Optimization of reactional temperature. (b) Optimization of reaction time.
(c) Optimization of X-gal concentration. (d) Detectable concentration of miR-17-5p.
The optimization experiment result for detection of miR-17-5p in cell free system showed that the best reaction conditions are 37 °C, 30 min and 60 µg/mL X-gal. The lowest visible detecting concentration of miR-17-5p is 5 pM.
Fig.16 The optimization of reactional conditions catalized by β-galactosidase in cell free system to increase the sensitivity of miR-146a-5p detection.
(a) Reaction temperature: 1. 27 °C; 2. 29 °C; 3. 31 °C; 4. 33 °C; 5. 35 °C; 6. 37 °C; 7. 39 °C; 8. 41 °C. (b) Reaction time: 1. 2 min; 2. 5 min; 3. 10 min; 4. 15 min; 5. 20 min; 6. 25min; 7. 30 min; 8. 35 min. (c) Concentration of X-gal: 1. 5 µg/mL; 2. 10 µg/mL; 3. 20 µg/mL; 4. 30 µg/mL; 5. 40 µg/mL; 6. 50 µg/mL; 7. 60 µg/mL; 8. 120 µg/mL. (d) Detectable concentration of miR-17-5p: 1. 50 fM; 2. 500 fM; 3. 5 pM; 4. 50 pM; 5. 500 pM; 6. 5 nM; 7. 50 nM; 8. 100 nM..
Fig.17 The quantitative analysis of conditional optimization for detection of miR-146a-5p using cell free system.
(a) Optimization of reactional temperature. (b) Optimization of reaction time.
(c) Optimization of X-gal concentration. (d) Detectable concentration of miR-146a-5p.
The optimization experiment result for detection of miR-146a-5p in cell free system showed that the best reaction conditions are 37 °C, 30 min and 60 µg/mL X-gal. The lowest visible detecting concentration of miR-146a-5p is 5 pM, similar with those for detection of miR-17-5p.
These experiments provided foundations for the subsequent experiment performed using cell free system on filter paper strip biosensor, which will be used in the detector to test microRNA concentration.
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3. Construction of filter paper biosensor
Filter paper is a ubiquitous, low-cost, easy to manufacture, store or transport biological analysis materials. After 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, forming a filter paper biosensor..
3.1 Preparation of three kinds of cell free expression systems extracted from BL21DlacZ strains
Three kinds of cell free systems were required to prepare. All of them were extracted from BL21DlacZ strains co-transformed with pET-28a-TorR-lacZ and pET-22b-TorT-TorS plasmids, transformed with pET-28a-miR-17-5p toehold switch-LacZ, and transformed with pET-28a-miR-146a-5p toehold switch-LacZ, respectively. All cell free systems were collected using lysis buffer and freeze-thaw method. The cell free expression systems were then prepared by mixing the cell extracts with other components such as ATP, phosphoenolpyruvate (PEP), amino acids, etc. (please refer to experiment section for details), and added X-gal to them.
3.2 Optimization of reaction conditions catalyzed by β-galactosidase in cell free expression systems on the filter paper strip biosensor
In order to increase the sensitivity and detecting efficiency, the reaction conditions catalyzed by β-galactosidase in cell free expression system on the paper strip biosensor were optimized under different temperature, reaction time, and X-gal concentration. The optimization results for detecting certain concentration of TMAO (100 µM), miR-17-5p (500 pM) and miR-146a-5p (500 pM) were showed in Fig.18-20, respectively.
Fig.18 The optimization result for detecting 100 µM TMAO in cell free system on the filter paper strip.
(a) Reaction temperature: 1. 17 °C; 2. 22 °C; 3. 27 °C; 4. 32 °C; 5. 37 °C. (b) Reaction time: 10 min; 2. 20 min; 3. 25 min; 4. 30 min; 5. 35 min. (c) Concentration of X-gal: 1. 10 µg/mL; 2. 20 µg/mL; 3. 30 µg/mL; 4. 40 µg/mL; 5. 50 µg/mL.
Fig.19 The optimization result for detecting 500 pM miR-17-5p in cell free system on the filter paper strip.
(a) Reaction temperature: 1. 22 °C; 2. 27 °C; 3. 32 °C; 4. 37 °C; 5. 40 °C. (b) Reaction time: 10 min; 2. 20 min; 3. 25 min; 4. 30 min; 5. 35 min. (c) Concentration of X-gal: 1. 10 µg/mL; 2. 20 µg/mL; 3. 30 µg/mL; 4. 40 µg/mL; 5. 50 µg/mL.
Fig.20 The optimization result for detecting 500 pM miR-146a-5p in cell free system on the filter paper strip.
(a) Reaction temperature: 1. 22 °C; 2. 27 °C; 3. 32 °C; 4. 37 °C; 5. 40 °C. (b) Reaction time: 10 min; 2. 20 min; 3. 25 min; 4. 30 min; 5. 35 min. (c) Concentration of X-gal: 1. 10 µg/mL; 2. 20 µg/mL; 3. 30 µg/mL; 4. 40 µg/mL; 5. 50 µg/mL.
To summarize, for detecting 100 µM TMAO in cell free expression system using filter paper strip biosensor, the optimized conditions are 37 °C, 30 min and 40 µg/mL X-gal. For detecting 500 pM miR-17-5p or miR-146a-5p in cell free expression system using filter paper strip biosensor, the optimized conditions are both 37 °C, 30 min and 50 µg/mL X-gal.
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3.3 Preparation of filter paper biosensor containing three kinds of cell free expression systems
According to the process mentioned above, three kinds of cell free expression systems were prepared and added X-gal to 40 µg/mL. After the filter paper was blocked with bovine serum albumin (BSA), washed and dried, each of the three cell free expression system was taken 5 µL volume to put onto the filter paper at different site, then freeze-dried to form a filter paper biosensor.
A certain concentration of TMAO (100 µM), miR-146a-5p (500 pM) and miR-17-5p (500 pM) were dropped onto the filter paper biosensor under 37 °C for 30 min, the result was shown in Fig.21. This biosensor is convenient for detecting TMAO, miR-17-5p, and miR-146a-5p from urine to screen AS at early stage.
Fig.21 Detecting TMAO, miR-146a-5p and miR-17-5p on the filter paper biosensor at the same time.
The labeled A, B, and C represent the results of detecting TMAO (100 µM), miR-146a-5p (500 pM), and miR-17-5p (500 pM), respectively.
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4. Conclusion
We constructed a filter paper biosensor which will be used in a detector to screen AS at early stage by detecting TMAO, miR-17-5p, and miR-146a-5p with urine sample. This filter paper biosensor used in a simple and convenient detector (please refer to our hardware) is a powerful tool to perform an early warning for AS, especially for asymptomatic AS. Due to the simplicity and portability, it is very beneficial for the elderly people to use it at home.
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5. Future plan and consideration
It was reported that the amount of TMAO produced in the human body depends on the individual dietary habits. Individuals with a preference for red meat, eggs, and fish produce a relatively high amount of TMAO, while those vegetarians have a lower content of TMAO. Therefore, in the future research, detection kits should be developed for individuals with different dietary habits, such as vegetarianism, mixed foods, and meat preference, to facilitate targeted detection of TMAO, monitor its development trends, and predict the occurrence of AS. Our project would be improved.
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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