Overview
In our project, we developed a biosensor to detect Shigella, Salmonella and Escherichia coli in refrigerator, based on nucleic acid aptamers, toehold switch and cell free hydrogel. These bacteria present in refrigerator are big threat to human health. If we can detect bacteria in the refrigerator in a timely manner and send out a warning, reminding people to clean and disinfect the refrigerator in time, it will be beneficial to protect people's physical health. Here, we show the main results of the experiments which are the basement of the biosensor constructed in our project.
1. Construction and identification of positive control plasmids
In order to make the detection of pathogenic bacteria more intuitive and visible, we used some chromoproteins as the reporters. We constructed 3 positive control plasmids containing 3 different chromoprotein genes.
One of them is amilCP gene (part K592009), of which protein shows purple color. The gene of amilCP was amplified using PCR method and inserted into the vector pET-28a (5362 bp). The construction of the plasmid pET-28a-amilCP was identified by restriction endonuclease digestion with EcoR I and Hind III according to the sequence. The length of amilCP gene is about 701 bp. Through agarose electrophoresis, we found that the lengths of digested fragments are consistent with the expected result (Fig. 1A).
In order to test whether the constructed plasmid pET-28a-amilCP express the chromoprotein, it was transformed into the BL21 strain. Then the transformed cells were plated on the agar. Some purple clones were shown in the plate (Fig. 1B), illustrating that the positive control plasmid pET-28a-amilCP was constructed well and expressed amilCP normally. It was used as positive control for the construction of subsequent RNA toehold switch plasmid.
Fig. 1 The identification of pET-28a-amilCP plasmid
(A) Electrophoresis result of plasmid digested by restriction endonucleases. M: Marker, 1: pET-28a-amilCP plasmid, 2: The plasmid digested by EcoR I and Hind III restriction endonucleases. (B) Expression of amilCP gene in BL21 cells transformed with pET-28a-amilCP plasmid.
Another reporter gene is mRFP (part J04450), of which protein shows red color. The gene of mRFP was amplified using PCR method and inserted into the expression vector pET-28a (5362 bp). The construction of the plasmid pET-28a-mRFP was also identified by restriction endonuclease digestion with EcoR I and Hind III. The length of mRFP gene is 714 bp. Through agarose electrophoresis, we found that the lengths of digested fragments are consistent with the expected result (Fig. 2A).
In order to test whether the constructed plasmid pET-28a-mRFP express the chromoprotein, it was transformed into the BL21 strain. Then the transformed cells were plated on the agar. Some red clones were shown in the plate (Fig. 2B), illustrating that the positive control plasmid pET-28a-mRFP was constructed well and expressed normally. It was used as positive control for the construction of subsequent RNA toehold switch plasmid.
Fig. 2 The identification of pET-28a-mRFP plasmid
(A) Electrophoresis result of plasmid digested by restriction endonucleases. M: Marker, 1: pET-28a-mRFP plasmid, 2: The plasmid digested by EcoR I and Hind III restriction endonucleases. (B) Expression of mRFP gene in BL21 cells transformed with pET-28a-mRFP plasmid.
The third reporter gene is EGFP (part E0040), of which protein shows florescent green color. The gene of EGFP was amplified using PCR method and inserted into the expression vector pET-28a (5362 bp). The construction of the plasmid pET-28a-EGFP was also identified by restriction endonuclease digestion with EcoR I and Hind III. The length of EGFP gene is 704 bp. Through agarose electrophoresis, we found that the lengths of digested fragments are consistent with the expected result (Fig. 3A).
In order to test whether the constructed plasmid pET-28a-EGFP express the florescent protein, it was transformed into the BL21 strain. Then the transformed cells were plated on the agar. Some green clones were shown in the plate (Fig. 3B), illustrating that the positive control plasmid pET-28a-EGFP was constructed well and expressed normally. It was used as positive control for the construction of subsequent RNA toehold switch plasmid.
Fig. 3 The identification of pET-28a-EGFP plasmid
(A) Electrophoresis result of plasmid digested by restriction endonucleases. M: Marker, 1: pET-28a-EGFP plasmid, 2: The plasmid digested by EcoR I and Hind III restriction endonucleases. (B) Expression of EGFP gene in BL21 cells transformed with pET-28a-EGFP plasmid.
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2. Construction and identification of aptamer toehold switch plasmids
According to the aptamer sequence of Shigella (please refer to Experiments section for details) and the construction rules of toehold switch, we designed the aptamer toehold switch with amilCP reporter gene, which was aimed to detect Shigella. The fragment of aptamer toehold switch was synthesized and inserted into pET-28a-amilCP plasmid using PCR method. This plasmid was named as pET-28a-Shig-amilCP which was identified by restriction endonucleases (EcoR I and Hind III) digestion assays. The inserted fragment length was about 801 bp. The agarose electrophoresis result showed that the inserted fragment length was consistent with the expected result (Fig. 4A).
Then the constructed plasmid was transformed into BL21 cells, and the result showed that no purple clone was observed (Fig. 4B), illustrating the aptamer toehold switch plasmid was constructed successfully, which purple protein didn’t express because of the translation inhibition by aptamer toehold switch.
Fig. 4 Identification of pET-28a-Shig-amilCP aptamer toehold switch plasmid.
(A) Electrophoresis result of plasmid and PCR. M: Marker, 1: Plasmid of pET-28a-Shig-amilCP, 2: The PCR result of aptamer toehold switch amilCP. (B) No purple clone was observed in BL21 cells transformed with pET-28a-Shig-amilCP plasmid.
According to the aptamer sequence of E. coli (please refer to Experiments section for details) and the construction rules of toehold switch, we designed the aptamer toehold switch with mRFP reporter gene, which was aimed to detect E. coli. The fragment of aptamer toehold switch was synthesized and inserted into pET-28a-mRFP plasmid using PCR method. This plasmid was named as pET-28a-Esch-mRFP which was identified by restriction endonucleases (EcoR I and Hind III) digestion assays. The inserted fragment length was about 850 bp. The agarose electrophoresis result showed that the inserted fragment length was consistent with the expected result (Fig. 5A).
Then the constructed plasmid was transformed into BL21 cells, and the result showed that no red clone was observed (Fig. 5B), illustrating the aptamer toehold switch plasmid was constructed successfully, which red protein didn’t express because of the translation inhibition by aptamer toehold switch.
Fig. 5 Identification of pET-28a-Esch-mRFP aptamer toehold switch plasmid.
(A) Electrophoresis result of plasmid and PCR. M: Marker, 1: Plasmid of pET-28a-Esch-mRFP, 2: The PCR result of aptamer toehold switch mRFP. (B) No purple clone was observed in BL21 cells transformed with pET-28a-Esch-mRFP plasmid.
According to the aptamer sequence of Salmonella (please refer to Experiments section for details) and the construction rules of toehold switch, we designed the aptamer toehold switch with EGFP reporter gene, which was aimed to detect Salmonella. The fragment of aptamer toehold switch was synthesized and inserted into pET-28a-EGFP plasmid using PCR method. This plasmid was named as pET-28a-Salm-EGFP which was identified by restriction endonucleases (EcoR I and Hind III) digestion. The inserted fragment length was about 794 bp. The agarose electrophoresis result showed that the inserted fragment length was consistent with the expected result (Fig. 6A).
Then the constructed plasmid was transformed into BL21 cells, and the result showed that no green clone was observed (Fig. 6B), illustrating the aptamer toehold switch plasmid was constructed successfully, which florescent protein didn’t express because of the translation inhibition by aptamer toehold switch.
Fig. 6 Identification of pET-28a-Salm-EGFP aptamer toehold switch plasmid.
(A) Electrophoresis result of plasmid and PCR. M: Marker, 1: Plasmid of pET-28a-Salm-EGFP, 2: The PCR result of aptamer toehold switch EGFP. (B) No green clone was observed in BL21 cells transformed with pET-28a-Salm-EGFP plasmid.
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3. Experiments of BL21 cells transformed with aptamer toehold switch plasmids
To study whether the aptamer toehold switches work or not, 3 complementary fragments of RNA aptamers of Shigella, E. coli and Salmonella were synthesized. Then both the aptamer toehold switch plasmids and their corresponding complementary fragments of RNA aptamers were co-transformed into the BL21 cells. The expression of reporter genes was observed (Fig.7), indicating that the aptamer toehold switches can control the expression of reporter proteins, and the complementary fragments of RNA aptamers can trigger the expression of corresponding reporter gene.
Fig.7 The expression of reporter genes in co-transformed cells.
(A) Reporter protein (amilCP) expressed in cells co-transformed with pET-28a-Shig-amilCP plasmid and corresponding complementary fragment of Shigella aptamer; (B)Reporter protein (EGFP) expressed in cells co-transformed with pET-28a-Salm-EGFP plasmid and corresponding complementary fragment of Salmonella aptamer; (C)Reporter protein (mRFP) expressed in cells co-transformed with pET-28a-Esch-mRFP plasmid and corresponding complementary fragment of E. coli aptamer.
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4. Optimization of reporter gene expression in cell free systems
To optimize the expression of reporter genes using the complementary fragments of aptamers and cell free systems which are used for making hydrogel to detect bacteria in fridge, we prepared the cell free systems extracted from BL21 cells transformed with constructed plasmids. After culturing the transformed cells for about 16 h, the cells were extracted to obtain cell extracts (please refer to Experiments section for details). Add some nutrient components to the cell extracts for getting cell free systems (please refer to Experiments section for details), then add the complementary fragments of aptamer of Shigella, E. coli and Salmonella to the corresponding 3 cell free systems, respectively.
The reaction conditions for expressing reporter proteins in cell free system were optimized using the synthesized complementary fragments of aptamers. The conditions including temperature, concentration of fragment, and reaction time were optimized in order to obtain sensitive and fast detection effects. The results of optimization experiment were showed in Fig. 8-Fig. 10.
Temperature is a primary factor affecting protein expression in vitro, and the optimal temperature for different proteins to express may be different. The optimization results showed that cell free system can express reporter proteins in a wide range of reaction temperatures, ranging from 4°C to 37°C, but the proper temperature varies from 25°C to 37°C for reporter proteins. The concentration of complementary fragment also has effect on reaction, and the lowest good concentration is about 2 uM. For visible reporter protein expression, it needed 6 h at least in cell free systems.
Fig. 8 The optimization results of reporter gene expression in cell free system transformed with pET-28a-Shig-amilCP after adding its corresponding complementary fragment of aptamer.
(A) temperature,1: 4℃, 2: 10℃, 3: 15℃, 4: 20℃, 5: 25℃, 6: 30℃, 7: 37℃; (B) concentration,1: 0.5uM, 2: 1uM, 3: 2uM, 4: 4uM, 5: 6uM, 6: 8uM, 7: 10uM; (C) reaction time, 1: 6h, 2: 8h, 3: 10h, 4: 13h, 5: 16h, 6: 20h, 7: 24h.
Fig.9 The optimization results of reporter gene expression in cell free system transformed with pET-28a-Esch-mRFP after adding its corresponding complementary fragment of aptamer.
(A) temperature,1: 4℃, 2: 10℃, 3: 15℃, 4: 20℃, 5: 25℃, 6: 30℃, 7: 37℃; (B) concentration,1: 0.5uM, 2: 1uM, 3: 2uM, 4: 4uM, 5: 6uM, 6: 8uM, 7: 10uM; (C) reaction time, 1: 6h, 2: 8h, 3: 10h, 4: 13h, 5: 16h, 6: 20h, 7: 24h.
Fig.10 The optimization results of reporter gene expression in cell free system transformed with pET-28a-Salm-EGFP after adding its corresponding complementary fragment of aptamer..
(A) temperature,1: 4℃, 2: 10℃, 3: 15℃, 4: 20℃, 5: 25℃, 6: 30℃, 7: 37℃; (B) concentration,1: 0.5uM, 2: 1uM, 3: 2uM, 4: 4uM, 5: 6uM, 6: 8uM, 7: 10uM; (C) reaction time, 1: 6h, 2: 8h, 3: 10h, 4: 13h, 5: 16h, 6: 20h, 7: 24h.
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5. Construction and identification of pET-28a-aptamer toehold LacZ plasmid
In order to improve sensitivity and reduce detection time, the report genes we used above were replaced with LacZ gene which can express an enzyme, amplifying the detection result. So, the pET-28a-Shig-lacZ, pET-28a-Esch-lacZ, and pET-28a-Salm-lacZ toehold switch plasmids were constructed using lacZ as reporter gene, which were used to detect Shigella, E. coli and Salmonella, respectively, in cell free systems. lacZ can express β-galactosidase, catalyzing the blue product generation using X-gal, which amplifies the detection results.
For identification of these plasmids, the restriction endonuclease digestion and PCR assay were performed. The results showed that the fragment lengths of aptamer toehold switch with lacZ were consistent with the expected results, indicating that pET-28a-Shig-LacZ, pET-28a-Esch-LacZ and pET-28a-Salm-LacZ plasmids were constructed successfully (Fig. 11 A, B and C).
Fig.11 Identification of aptamer toehold switch with LacZ gene plasmids.
(A) pET-28a-Shig-LacZ plasmid. M: Marker, 1: The plasmid of pET-28a-Shig-LacZ, 2: PCR result, 3: Digestion result by EcoR I and Hind III restriction endonuclease. (B) pET-28a-Esch-LacZ plasmid. M: Marker, 1: The plasmid of pET-28a-Esch-LacZ, 2: PCR result, 3: Digestion result by EcoR I and Hind III restriction endonuclease. (C) pET-28a-Salm-LacZ plasmid. M: Marker, 1: The plasmid of pET-28a-Salm-LacZ, 2: PCR result, 3: Digestion result by EcoR I and Hind III restriction endonuclease.
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6. Construction of BL21 cells lacking of LacZ gene
In order to eliminate the influence of the BL21 cells’ own β-galactosidase, 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 (1.6 kb) was obtained by PCR amplification (Fig. 12). This PCR product of kanamycin gene was flanked with the start and end sequences of LacZ gene.
Fig. 12 The PCR result of Kanamycin gene using pET-28a vector as a template.
M: Marker, 1: Kanamycin gene amplified by PCR method.
The PCR product of amplified kanamycin gene flanked with the start and end sequences of LacZ gene was transformed into BL21 strain in which homologous recombination occurred between the PCR product and the LacZ gene of BL21 genome, and BL21 mutant strain with LacZ gene deletion (BL21DLacZ) was obtained. This BL21DLacZ strain was screened out with kanamycin and confirmed by PCR results of LacZ and kanamycin genes (Fig. 13), indicating that the LacZ gene deletion BL21 strain (BL21DLacZ) with kanamycin resistant was constructed successfully.
Fig. 13 The PCR results of Kanamycin and LacZ genes in kanamycin resistant BL21DLacZ strain.
M: Marker, 1: The amplification of Kanamycin gene, 2: The amplification of LacZ gene (No band).
To identify whether the LacZ gene of recombinant plasmid express or not，both the complementary fragment of RNA aptamer and the plasmid pET-28a-Esch-lacZ were transformed into BL21DLacZ strain. After culture 12 h with fresh LB medium containing Kanamycin and Ampicillin, the LacZ protein (β-galactosidase) was purified using 6x His tag column for identification. The SDS-PAGE electrophoresis result was shown in Fig.14.
Fig. 14 The SDS-PAGE result shows expression and purification of LacZ protein.
M: Marker, 1: All supernatant proteins of BL21DLacZ strain, 2: All supernatant proteins of BL21DLacZ strain transformed with pET-28a-Esch-LacZ and the complementary fragment of E. coli aptamer, 3: Purified LacZ protein from the supernatant proteins of sample 2.
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7. Experiment performed in cell free system using LacZ gene as reporter
Due to concerns about the pathogenicity and safety, we did not conduct the detection experiments using bacteria. We only used the complementary fragment of E. coli aptamer to show the feasibility of our constructed cell free system containing pET-28a-Esch-LacZ plasmid for bacteria detection. The cell free system was prepared using plasmid pET-28a-Esch-LacZ transformed BL21DLacZ strain. Complementary fragment and X-gal (substrate of β-galactosidase) were added to the cell free system under different conditions including temperature, concentration of complementary fragment, concentration of X-gal, and detection time. Since the maximum absorbance of the blue product is located at 615 nm, the experiment results were quantified using absorbance value, which were showed in Fig. 15. The results indicated that the cell free system works well. For intuitive and visible good result, the best conditions for reaction are 20℃, 0.5 uM complementary fragment of aptamer, 100 ug/mL X-gal, and 1 h reaction time.
Fig.15 The quantification result of experiment performed in cell free system containing pET-28a-Esch-LacZ plasmid under different conditions.
(A) Absorbance of the end product catalyzed by β-galactosidase expressed in cell free system with 1 uM complementary fragment of aptamer and 100 ug/mL X-gal, 1 h reaction time, under different temperature; (B) Absorbance of the end product catalyzed by β-galactosidase expressed in cell free system with 100 ug/mL X-gal and different concentration of complementary fragment of aptamer,1 h reaction time, under 20℃; (C) Absorbance of the end product catalyzed by β-galactosidase expressed in cell free system with1 uMcomplementary fragment of aptamer and different concentration of X-gal, 1 h reaction time, under 20℃; (D) Absorbance of the end product catalyzed by β-galactosidase expressed in cell free system with 1 uM complementary fragment of aptamer and 100 ug/mL X-gal, different reaction time, under 20℃.
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8. Experiment on cell free system hydrogel
Hydrogel is a three-dimensional (3D) network polymer with hydrophilic groups, which can absorb moisture but are insoluble in water. Hydrogel is widely used in various fields due to its unique physical and chemical properties. It is suitable for the three-dimensional structure of cell growth and differentiation. In addition, hydrogel has good mechanical property.
In our experiment, we combined the cell free system with methacrylated gelatin (GelMA) hydrogel which has excellent biocompatibility and cell reaction characteristics, making high expression ability of cell free system and controllable catalyzing reaction. This combination made the possibility of bacteria detection in fridge using cell free system. So, the cell free system hydrogel was constructed using cell free system extracted from BL21DLacZ strain transformed with pET-28a-Esch-LacZ plasmid and hydrogel ( GelMA).
Add 0.5 uM or 1 uM complementary fragment of aptamer and 100 ug/mL X-gal onto the GelMA hydrogel containing cell free system, reaction 2 h under 4℃. The result was showed in Fig. 16, indicating that it is possible using the cell free system hydrogel to detect bacteria in refrigerator.
Fig. 16 Experiment on cell free system hydrogel, reaction 2 h, under 4℃.
(A) The negative control without complementary fragment of aptamer or X-gal; (B) The result using 0.5 uM complementary fragment of aptamer and 100 ug/mL X-gal; (C) The result using 1 uM complementary fragment of aptamer and 100 ug/mL X-gal.
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9. Conclusion
We constructed a biosensor using cell free system hydrogel containing RNA toehold switch, which is the main component of a detector used for detecting pathogenic bacteria in refrigerator. When the bacteria fall to the hydrogel in fridge, it will switch on the RNA toehold to express lacZ gene and produce β-galactosidase, catalyzing the blue product which is detected by the biosensor. This information is scanned and transferred by RGB color sensor to the pilot lamp, making the red LED light up on the screen of refrigerator door, reminding people to clean or disinfect the refrigerator in time. It is very convenient and beneficial for the people to use it in refrigerator at home.
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10. Future plan and consideration
Due to the low temperature of the refrigerator, the enzymatic reaction occurring on the biosensor is relatively slow. Therefore, in future research and development, we will continuously optimize the conditions of enzymatic reaction to increase enzyme activity, achieving better detection results. Or we may search for an enzyme that can tolerate low temperature, to instead the current enzyme (β-galactosidase).
In addition, it is necessary to continuously increase the number of bacteria aptamers for detecting more pathogens and broadening the bacteria range of detection.
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 in the future.
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