Overview
In this section, we would like to demonstrate our engineering design cycle to show the engineering success and effort. Also, we constructed some plasmids which are involved in our project in 2024 competition season. Here, we would like to show the construction of a new part (BBa_K5074001).
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1. The engineering design cycle
1.1 Introduction
To establish a biosensor for detecting bacteria in refrigerator, we need to construct some recombinant plasmids containing RNA aptamer toehold switches, which can recognize and bind to some bacteria to trigger the expression of reporter genes. We used 3 reporter genes amilCP, mRFP and EGFP to construct 3 recombinant plasmids which are used to detect 3 bacteria Shigella, Escherichia coli and Salmonella, respectively. Here, we took the construction of recombinant plasmid for detecting Escherichia coli (E.coli) as an example to demonstrate the engineering success.
1.2 Engineering cycle
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1.2.1 Design Stage |
According to the aptamer sequence of E.coli and the construction rules of toehold switch, we designed the aptamer toehold switch with mRFP reporter gene which was aimed to detect E.coli. At the presence of E.coli or the complementary fragment of aptamer (trigger RNA), it can bind to the toehold switch and trigger the expression of reporter gene (Fig.1).
Fig.1 The construction principle of RNA Toehold Switch using mRFP reporter gene.
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1.2.2 Build Stage |
The sequence 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. After the construction, it was identified using PCR amplification. The inserted fragment length was about 850 bp. The agarose electrophoresis result showed that the PCR result was consistent with the expected length (Fig.2).
Fig.2 Electrophoresis Identification of pET-28a-Esch-mRFP aptamer toehold switch plasmid and PCR amplification.
M: Marker, 1: Plasmid of pET-28a-Esch-mRFP, 2: The PCR result of aptamer toehold switch mRFP.
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1.2.3 Test Stage |
Then the constructed plasmid was transformed into BL21 cells, and the result showed that no red clone was observed (Fig.3A), 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.3 The plasmid pET-28a-Esch-mRFP was transformed into BL21 cells, no red clone was observed, compared to the BL21 cells transformed with pET-28a-mRFP plasmid.
(A) BL21 cells transformed with pET-28a-Esch-mRFP plasmid; (B) BL21 cells transformed with pET-28a-mRFP plasmid.
To study whether the E.coli aptamer toehold switch work or not, the complementary fragment of RNA aptamer of E.coli was synthesized. Then both the complementary fragment of RNA aptamer and the plasmid pET-28a-Esch-mRFP were transformed into BL21 cells. The expression of reporter gene mRFP was observed (Fig.4), indicating that the aptamer toehold switch can control the expression of reporter gene, and the complementary fragment of RNA aptamer can trigger the expression of reporter gene.
Fig.4 After transformation of both complementary fragment of RNA aptamer and the pET-28a-Esch-mRFP plasmid, the expression of reporter gene mRFP was observed.
To optimize the expression of reporter gene mRFP using the complementary fragment of aptamer and cell free system which is used for making hydrogel to detect E.coli in fridge, we prepared the cell free system extracted from BL21 cells transformed with pET-28a-Esch-mRFP plasmid. Add the complementary fragment of aptamer to the cell free system to observe the reporter gene expression.
The reaction conditions for expressing reporter protein in cell free system were optimized. The conditions including temperature, concentration of fragment, and reaction time were optimized in order to obtain sensitive and fast detection effect.
The result (Fig.5) shows that cell free system can express reporter protein ranging from 4°C to 37°C, but the proper temperature varies from 25°C to 37°C for mRFP expression. The lowest good concentration is about 2 uM. For visible reporter protein expression, it needed 6 h at least in cell free systems.
Fig.5 The optimization result of reporter gene expression in cell free system transformed with pET-28a-Esch-mRFP after adding the complementary fragment of aptamer.
(A) temperature,1: 4℃, 2: 10℃, 3: 15℃, 4: 20℃, 5: 25℃, 6: 30℃, 7: 37℃; (B) concentration,1: 0.5 uM, 2: 1 uM, 3: 2 uM, 4: 4 uM, 5: 6 uM, 6: 8 uM, 7: 10 uM; (C) reaction time, 1: 6 h, 2: 8 h, 3: 10 h, 4: 13 h, 5: 16 h, 6: 20 h, 7: 24 h.
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1.2.4 Learn Stage |
We successfully constructed the plasmid pET-28a-Esch-mRFP, which can express reporter gene mRFP under the control of complementary fragment of E.coli aptamer. At the presence of complementary fragment of E.coli aptamer, mRFP was expressed well. But from the optimization result, we figured out that to express reporter gene mRFP in cell free system, 2 uM complementary fragment of aptamer is relatively high which is not sensitive, and it needs long reaction time (6 h) for detection. Both of which required to improve.
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1.2.5 Re-Design |
In order to improve sensitivity and reduce detection time, the report gene was replaced with LacZ gene which can express an enzyme, amplifying the detection result. So, the pET-28a-Esch-lacZ toehold switch plasmid was constructed using lacZ as reporter gene, used to detect E. coli in cell free systems. lacZ can express β-galactosidase, catalyzing the blue product generation using X-gal, which amplifies the detection signals (Fig.6).
Fig.6 The construction principle of RNA Toehold Switch using LacZ reporter gene.
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1.2.6 Re-Builed |
For identification of reconstructed plasmid pET-28a-Esch-lacZ, 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-Esch-LacZ was constructed successfully (Fig.7).
Fig.7 Identification of aptamer toehold switch plasmid pET-28a-Esch-LacZ.
M: Marker, 1: The plasmid of pET-28a-Esch-LacZ, 2: PCR result, 3: Digestion result by EcoR I and Hind III restriction endonuclease.
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1.2.7 Re-Test |
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 (with LacZ deletion). 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.8.
Fig. 8 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.
To test the feasibility of our constructed cell free system containing pET-28a-Esch-LacZ plasmid for bacteria detection. The cell free system was prepared using pET-28a-Esch-LacZ plasmid 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.9.
Fig.9 The quantification results 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 with 1 uM complementary 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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1.2.8 Re-Learn |
The quantification results of Fig.9 indicated that the cell free system extracted from pET-28a-Esch-LacZ transformed BL21 cells 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, all of which are improved.
Summary:
Through two engineering cycles, we constructed successfully pET-28a-Esch-LacZ plasmid which is triggered to effectively express reporter gene by the complementary fragment of aptamer. This plasmid will be used to detect E. coli in the future.
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2. Construction of a new part BBa_K5074001
In this competition season, we also constructed a new part (BBa_K5074001). It is an AmilCP generator controlled by Shigella RNA aptamer toehold switch. At the presence of Shigella dysenteriae, it binds to its RNA aptamer, opening the toehold switch to trigger the expression of amilCP, which is easily detected. It is used to detect the bacteria Shigella in refrigerator.
This part comprises Shigella dysenteriae RNA aptamer, RBS (B0034), Linker and reporter gene amilCP (K592009). Its sequence was analyzed using SnapGene Viewer for restriction enzyme cleaving sites to construct the plasmid pET-28a-Shig-amilCP using the proper enzymes. The analysis result with SnapGene Viewer was shown in Fig.10.
Fig.10 The endonuclease map of BBa_K5074001 analyzed using SnapGene Viewer, showing the restriction enzyme information (no EcoRI and Hind III cleaving sites).
The sequence 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 PCR amplification. The inserted fragment length was about 801 bp. The agarose electrophoresis result showed that the PCR product was consistent with the expected result (Fig.11).
Fig.11 Electrophoresis Identification of pET-28a-Shig-amilCP aptamer toehold switch plasmid and PCR amplification.
M: Marker, 1: Plasmid of pET-28a-Shig-amilCP, 2: The PCR result of aptamer toehold switch amilCP.
Then the constructed plasmid was transformed into BL21 cells, and the result showed that no purple clone was observed (Fig.12A), 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.12 The plasmid pET-28a-Shig-amilCP was transformed into BL21 cells, no purple clone was observed, compared to the BL21 cells transformed with pET-28a-amilCP plasmid.
(A) BL21 cells transformed with pET-28a-Shig-amilCP plasmid; (B) BL21 cells transformed with pET-28a-amilCP plasmid.
To study whether the Shigella aptamer toehold switch work or not, the complementary fragment of RNA aptamer of Shigella was synthesized. Then both the complementary fragment of RNA aptamer and the plasmid pET-28a-Shig-amilCP were transformed into BL21 cells. The expression of reporter gene amilCP was observed (Fig.13), indicating that the aptamer toehold switch can control the expression of reporter gene, and the complementary fragment of RNA aptamer can trigger the expression of reporter gene.
Fig.13 After transformation of both complementary fragment of RNA aptamer and the pET-28a-Shig-amilCP plasmid, the expression of reporter gene amilCP was observed.
To optimize the expression of reporter gene amilCP using the complementary fragment of aptamer and cell free system which is used for making hydrogel to detect Shigella in fridge, we prepared the cell free system extracted from BL21 cells transformed with pET-28a-Shig-amilCP plasmid. Add the complementary fragment of aptamer to the cell free system to observe the reporter gene expression.
The reaction conditions for expressing reporter protein in cell free system were optimized. The conditions including temperature, concentration of fragment, and reaction time were optimized in order to obtain sensitive and fast detection effect.
The result of optimization experiment was shown in Fig.14. The result shows that cell free system can express reporter protein in a wide range of temperatures, ranging from 4°C to 37°C, but the proper temperature varies from 25°C to 37°C for amilCP expression. 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.14 The optimization result of reporter gene expression in cell free system transformed with pET-28a-Shig-amilCP after adding the complementary fragment of aptamer.
(A) temperature,1: 4℃, 2: 10℃, 3: 15℃, 4: 20℃, 5: 25℃, 6:30℃, 7:37℃; (B) concentration,1: 0.5 uM, 2: 1 uM, 3: 2 uM, 4: 4 uM, 5: 6 uM, 6:8 uM, 7: 10 uM; (C) reaction time, 1:6 h, 2: 8 h, 3: 10 h, 4: 13 h, 5: 16 h, 6: 20 h, 7: 24 h.
Summary:
We successfully constructed the plasmid pET-28a-Shig-amilCP, which can express reporter gene amilCP under the control of complementary fragment of Shigella aptamer. At the presence of complementary fragment of Shigella aptamer, amilCP was expressed well. The optimized conditions to express reporter gene in cell free system are 25℃, 2 uM complementary fragment of aptamer, at least reaction 6 h.
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