1. We created a new part BBa_K4167000
BBa_K4167000 part is a standard part, in which toehold switch-amilCP sequence was inserted into pSB1C3 plasmid. It was designed to express amilCP protein triggered by miRNA 34a-5p. This part is used to detect the amount of miRNA 34a-5p in samples.
To construct the standard part, toehold switch-amilCP was synthesized and checked the restriction enzyme information, which is shown as follows (Fig.1).
Fig.1 The map of toehold switch-amilCP described with SnapGene Viewer, showing the restriction enzyme information (no EcoRI and PstI sites).
After detecting the restriction enzyme information of toehold switch-amilCP using SnapGene software, it was inserted into the pSB1C3 plasmid to construct the standard part pSB1C3-toehold switch-amilCP with PCR method. Then it was identified as follows (Fig.2).
Fig.2 Identification of standard part pSB1C3-toehold switch-amilCP using PCR and digestion with EcoRI and PstI.
M: Marker; 1: PCR result; Digestion result.
Toehold switch-amilCP plasmid is designed to express the amilCP protein controlled by the toehold switch and miRNA 34a-5p. It comprises the antisense sequence of miRNA 34a-5p, RBS, Linker and part sequence of miRNA 34a-5p, which form a toehold switch, as well as the gene of marker protein amilCP. At the presence of miRNA 34a-5p, it binds to its antisense sequence, opening the toehold switch to trigger the expression of amilCP, which is easily measured. The mechanism is shown as Fig.3.
Fig.3 The mechanism of toehold switch-amilCP.
Toehold switch-amilCP was also cloned into pET-28a expression vector, constructing the recombined plasmid pET-28a-toehold switch-amilCP. After it was transfected into BL21 strain, no amilCP protein (purple color) could be observed with naked eyes, indicating that the toehold switch was effective. However, after transfection with miRNA 34a-5p into the BL21 strain transfected with pET-28a-toehold switch-amilCP, some transfected clones appeared purple color, which were shown in Fig.4.
Fig.4 The effectiveness of toehold switch-amilCP.
Note: Bacteria clones only transfected with toehold switch-amilCP appeared white color, while bacteria clones transfected with both toehold switch-amilCP and miRNA 34a-5p appeared purple color (miRNA 34a-5p switched on the expression of amilCP).
To increase the yielding of marker protein amilCP, some different culture conditions were optimized, including the pH value, temperature, fermentation time, and the concentration of transfected miRNA. BL21 strain containing toehold switch plasmid were cultured under different conditions. Since reporter protein amilCP has color, we can easily intuitively find the optimal conditions through the change of color. 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.
Fig.5 Optimization of culture conditions of BL21 strain with toehold switch-amilCP plasmid and miRNA 34a-5p.
2. We created a 3D hardware to show how our detector works
2.1 Background
Our diagnostic solution is based on color change of paper strip sensor for detecting miRNA and for this reason we created a acquire image hardware design. At present, nucleic acid amplifier can be used to detect miRNA by PCR method. However, the operation is complex, which needs high requirements for personnel, environment and equipment, and the cost is high.
2.2 Introduction
In order to facilitate and quickly obtain diagnostic tests’ results, we created a small device for it (Fig.6). There is a window on the side of the device where two paper strips can be placed. One paper strip is for reference and the other is for sample detection, which is convenient for qualitative and quantitative analysis. There is a cell phone rack on the device, which can be retracted to facilitate the placement of smart phones of different sizes and specifications. The smart phone is used for acquiring image. Appropriate light sources are installed inside the device to provide sufficient brightness, and different light sources are also provided to stimulate reporter proteins of different colors.
Fig.6 3D-printed device for results analysis from biosensors
2.3 3D model
In order to make the device more clearly acquire the markers of different colors, we add a filter on the device and place it on the rack above the paper strip (Fig.7).
Fig.7 3D-printed device and filter for results analysis from biosensors
2.4 Combination
Paper strip sensor, filter and the device are shown in Fig.8, which is convenient for obtain diagnostic tests’ results.
Fig.8 3D-printed device, filter, and paper strip sensor
2.5 Hardware characteristics
Based on different requirement of emission, an airtight environment is necessary for high quality images. The 3D printed black case ensures the correct location of the filters and reproducible conditions in terms of distance and darkness for the imaging process.
Exact positioning of the filters in front of the camera and the flash is important in order to generate images appropriate for detection.
The top smartphone inlay can be specifically adapted to different smartphone types. A smartphone application was needed. With the help of this application fixed points on the images can be chosen and the median value of the paper strip can be analyzed. Considering control reactions, the app calculates whether a contamination is present.
The paper strip can be placed on the push loading drawer and be inserted into the box.
2.6 Further modifications
For better transportation and safer usage each spot could have a glued clear plastic bottom.
3. References
[1]. Wan Y, Liu Y, Wang X, et al. Identification of differential microRNAs in cerebrospinal fluid and serum of patients with major depressive disorder. PLoS One, 2015 Mar 12;10(3): e0121975. doi: 10.1371/journal.pone.0121975 [2]. Zhou L, Zhu Y, Chen W, et al. Emerging role of microRNAs in major depressive disorder and its implication on diagnosis and therapeutic response. J Affect Disord. 2021;286: 80-86. doi: 10.1016/j.jad.2021.02.063 [3]. Green AA, Silver PA, Collins JJ, et al. Toehold switches: de-novo-designed regulators of gene expression. Cell. 2014;159(4):925-39. doi: 10.1016/j.cell.2014.10.002