iGEM 2022 — Team icjfls

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{% block title %}Design{% endblock %}
{% block lead %}**We Designed toehold switch to detect miRNA for diagnosis of MDD.**{% endblock %}
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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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{% block title %}Engineering Success{% endblock %}
{% block lead %}**We constructed 2 parts successfully.**{% endblock %}
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In this part, we want to show engineering success by demonstration the design and construction of 2 new parts. Both of them worked as expected:

## 1. [BBa\_K4167001](http://parts.igem.org/Part%3ABBa_K4167001)

---

BBa\_K4167001 part is a standard part, in which toehold switch-mRFP sequence was inserted into pSB1C3 plasmid. It was designed to express mRFP protein triggered by miRNA 221-3p. It is used to detect the amount of miRNA 221-3p in samples.

To construct the standard part, toehold switch-mRFP was synthesized and checked the restriction enzyme information, which is shown as follows(Fig.1).

![](https://static.igem.wiki/teams/4167/wiki/fig-1-the-map-of-toehold-switch-mrfp-described-with-snapgene-viewer-showing-the-restriction-enzyme-information-no-ecori-and-psti-sites.jpg)

**Fig.1 The map of toehold switch-mRFP described with SnapGene Viewer, showing the restriction enzyme informatio**n (no *Eco*RI and *Pst*I sites).

After detecting the restriction enzyme information of toehold switch-mRFP using SnapGene software, it was inserted into the pSB1C3 plasmid to construct the standard part pSB1C3-toehold switch-mRFP with PCR method. Then it was identified as follows (Fig.2).

![](https://static.igem.wiki/teams/4167/wiki/fig-2-identification-of-standard-part-psb1c3-toehold-switch-mrfp-using-pcr-and-digestion-with-ecori-and-psti.jpg)

**Fig.2 Identification of standard part pSB1C3-toehold switch-mRFP using PCR and digestion with *Eco*RI and *Pst*I.**
M: Marker; 1: PCR result; Digestion result.

Toehold switch-mRFP plasmid is designed to express the mRFP protein controlled by the toehold switch and miRNA 221-3p. It comprises the antisense sequence of miRNA 221-3p, RBS, Linker and part sequence of miRNA 221-3p, which form a toehold switch, as well as the gene of marker protein mRFP. At the presence of miRNA 221-3p, it binds to its antisense sequence, opening the toehold switch to trigger the expression of mRFP, which is easily measured. The mechanism is shown as Fig.3.

![](https://static.igem.wiki/teams/4167/wiki/fig-3-the-mechanism-of-toehold-switch-mrfp.jpg)

**Fig.3 The mechanism of toehold switch-mRFP.**

Toehold switch-mRFP was also cloned into pET-28a expression vector, constructing the recombined plasmid pET-28a-toehold switch-mRFP. After it was transfected into BL21 strain, no mRFP protein (red color) could be observed with naked eyes, indicating that the toehold switch was effective. However, after transfection with miRNA 221-3p into the BL21 strain transfected with pET-28a-toehold switch-mRFP, some transfected clones appeared red color, which were shown in Fig.4.

![](https://static.igem.wiki/teams/4167/wiki/fig-4-the-effectiveness-of-toehold-switch-mrfp.jpg)

**Fig.4 The effectiveness of toehold switch-mRFP.**
Bacteria clones only transfected with toehold switch-mRFP appeared white color, while bacteria clones transfected with both toehold switch-mRFP and miRNA 221-3p appeared red color (miRNA 221-3p switched on the expression of mRFP).

To increase the yielding of marker protein mRFP, 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 mRFP 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*.

![](https://static.igem.wiki/teams/4167/wiki/fig-5-optimization-of-culture-conditions-of-bl21-strain-with-toehold-switch-mrfp-plasmid-and-mirna221-3p.jpg)

**Fig.5 Optimization of culture conditions of BL21 strain with toehold switch-mRFP plasmid and miRNA221-3p.**

All these figures showed above indicated that the part BBa\_K4167001 worked as expected.

## 2. [BBa\_K4167002](http://parts.igem.org/Part%3ABBa_K4167002)

---

BBa\_K4167002 part is a standard part, in which toehold switch-amilGFP sequence was inserted into pSB1C3 plasmid. It was designed to express amilGFP protein triggered by miRNA let-7d-3p. It is used to detect the amount of miRNA let-7d-3p in samples.

To construct the standard part, toehold switch-amilGFP was synthesized and checked the restriction enzyme information, which is shown as follows (Fig.6).

![](https://static.igem.wiki/teams/4167/wiki/fig-6-the-map-of-toehold-switch-amilgfp-described-with-snapgene-viewer.jpg)

**Fig.6 The map of toehold switch-amilGFP described with SnapGene Viewer, showing the restriction enzyme information** (no *Eco*RI and *Pst*I sites).

After detecting the restriction enzyme information of toehold switch-amilGFP using SnapGene software, it was inserted into the pSB1C3 plasmid to construct the standard part pSB1C3-toehold switch-amilGFP with PCR method. Then it was identified as follows (Fig.7).

![](https://static.igem.wiki/teams/4167/wiki/fig-7-identification-of-standard-part-psb1c3-toehold-switch-amilgfp-using-pcr-and-digestion-with-ecori-and-psti.jpg)

**Fig.7 Identification of standard part pSB1C3-toehold switch-amilGFP using PCR and digestion with *Eco*RI and *Pst*I.**
M: Marker; 1: PCR result; Digestion result.

Toehold switch-amilGFP plasmid is designed to express the amilGFP protein controlled by the toehold switch and miRNA let-7d-3p. It comprises the antisense sequence of miRNA let-7d-3p, RBS, Linker and part sequence of miRNA let-7d-3p, which form a toehold switch, as well as the gene of marker protein amilGFP. At the presence of miRNA let-7d-3p, it binds to its antisense sequence, opening the toehold switch to trigger the expression of amilGFP, which is easily measured. The mechanism is shown as Fig.8.

![](https://static.igem.wiki/teams/4167/wiki/fig-8-the-mechanism-of-toehold-switch-amilgfp.jpg)

**Fig.8 The mechanism of toehold switch-amilGFP.**

Toehold switch-amilGFP was also cloned into pET-28a expression vector, constructing the recombined plasmid pET-28a-toehold switch-amilGFP. After it was transfected into BL21 strain, no amilGFP protein (yellow color) could be observed with naked eyes, indicating that the toehold switch was effective. However, after transfection with miRNA let-7d-3p into the BL21 strain transfected with pET-28a-toehold switch-amilGFP, some transfected clones appeared yellow color, which were shown in Fig.9.

![](https://static.igem.wiki/teams/4167/wiki/fig-9-the-effectiveness-of-toehold-switch-amilgfp.jpg)

**Fig.9 The effectiveness of toehold switch-amilGFP.**
Bacteria clones only transfected with toehold switch-amilGFP appeared white color, while bacteria clones transfected with both toehold switch-amilGFP and miRNA let-7d-3p appeared yellow color (miRNA let-7d-3p switched on the expression of amilGFP).

To increase the yielding of marker protein amilGFP, 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 amilGFP 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.*

![](https://static.igem.wiki/teams/4167/wiki/fig-10-optimization-of-culture-conditions-of-bl21-strain-with-toehold-switch-amilgfp-plasmid-and-mirna-let-7d-3p.jpg)

**Fig.10 Optimization of culture conditions of BL21 strain with toehold switch-amilGFP plasmid and miRNA let-7d-3p.**

All these figures showed above indicated that the part BBa\_K4167002 worked as expected.

## 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
```

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}**We describe the research, experiments, and protocols in our iGEM project.**{% endblock %}
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## Overview

---

In our project, we established a major depression disorder (MDD) diagnosis platform to detect microRNAs (miRNAs). We developed a paper strip sensor to detect miRNA based on toehold switch and cell free expression system, which facilitate the intuitive detection for MDD at remote areas or resource limited environments or even at home. The experimental protocals are shown as follows.

## The Competent Cell Preparation

---

1. Streak the *E. coli* stock on a LB-agar plate. Incubate the plate at 37°C overnight.
2. Pick a single well-isolated colony and inoculate it into 3mL of LB broth solution. Incubate it at 37°C overnight (more than 16 h) with shaking at 220 rpm.
3. Transfer 250uL of the saturated overnight solution to a sterile 50mL polypropylene tube containing 25mL of LB medium. Incubate the *E. coli* at 37°C with shaking at 220 rpm until OD600 reaching 0.6. This usually takes 2.5 h. Check the OD every 30 min after 1 h to avoid overgrowth.
4. When the medium reaches an OD600 of 0.6, chill the tube on the ice for 30 min and then centrifuge at 8000rpm for 1min at 4°C, discard the supernatant.
5. Re-suspend the *E. coli* in 2.5mL of ice-cold TSS solution with gentle swirling. Incubate on ice for 10 min. Aliquot in 50uL per tube. Now the competent cells are ready to be transformed.
Note: Transformation and Storage Solution (TSS) contains LB medium 85% 8.5mL, PEG (wt/vol, Mw 8000) 10% 1.0g, DMSO (vol/vol) 5% 500μL, and MgCl2 (pH 6.5) 50mM 500μL.

## Plasmid Transforming and Pick monoclonal

---

1. Thaw TSS cells on ice.
2. Add 5uL Synthetic plasmid DNA in TSS cells, pipette gently to mix.
3. Incubate it on ice for 30 min with occasional mixing.
4. Heat shock at 42°C for 90 sec.
5. After heat shock, put it on ice for 2 min, add 0.8mL LB medium into the cells.
6. Shake and incubate at 37°C for 60 min at 120 rpm.
7. Take out 100uL medium and spread them on the appropriate agar plates with certain antibiotic.
8. Incubate the plates at 37°C overnight.
9. Pick monoclonal into certain antibiotic LB medium, and incubate the medium at 37°C overnight.

## Plasmid Isolation Protocol

---

1. The bacteria that contained target plasmid were cultivated at 37°C overnight.
2. Take out 1,500uL solution of cultivated bacteria to a 2mL micro-centrifuge tube. The tube was centrifuged at 12,000rpm for 1 min at room temperature, and then the supernatant was discarded. In this tube, repeating these steps three times to increase the concentration of the bacteria.
3. Add 250uL Solution I buffer to suspend the precipitation. Complete suspension is vital for obtaining good plasmid yield by vortexing or pipetting up and down.
4. Add 250uL Solution II buffer, mix gently upside down 4-6 times to make a cleared lysate. A 2-3 min incubation at room temperature may be necessary. However, this step should not be more than 5 min, and avoid vigorous mixing as doing so will shear chromosomal DNA and lower plasmid purity.
5. Add 350uL Solution III buffer, mix gently and fully upside down 6-8 times, centrifuge the tubes at 12,000rpm for 10 min at room temperature.
6. Put a HiBind DNA Mini Column into a 2mL Collection Tube.
7. The supernatant in step 4 is transferred to the Mini Column, and centrifuged at 12,000rpm for 1 min, the filtrate is discarded.
8. Put the Column back into the Collection Tube, add 500uL HBC Buffer, centrifuge at 12,000rpm for 1 min, the filtrate is discarded.
9. Put the Column back into the Collection Tube, add 700uL Washing buffer, centrifuge at 12,000rpm for 1 min, the filtrate is discarded. Repeat the washing step once.
10. Put the Column back into the Collection Tube and centrifuge at 12,000 rpm for 2 min.
11. Put the Column into a new 1.5mL micro-centrifuge tube and leave it for 2 min at room temperature. Then add 50uL ddH2O to the center of the Column matrix, and leave it at room temperature for 1 min. Centrifuge at12,000 rpm for 1 min. Discard the HiBind DNA Mini Column.
12. Measure concentration of extracted plasmid DNA by nanodrop 1000, and store plasmid DNA at -20°C.

## Plasmid DNA Restriction endonuclease Digestion

---

DNA for downstream applications is usually digested with restriction endonucleases. Type II restriction enzymes are the most widely used in molecular biology application.
The reaction volume are carried out in 20µL.
1. Add components to a clean tube in the order as follow:
    1 µL DNA (1 µg/µL)
    2 µL 10x buffer
    1 µL each restriction enzyme (*EcoR1* and *HindⅢ*)
    15 µL ddH2O
2. Mix gently and fully, and incubate the tube at certain temperature (37°C) for 1 h.
3. Stop the reaction by heat inactivation (65°C for 15 min).
4. The digested DNA is ready for use in research applications.

## PCR protocol

---

In the PCR protocol, reaction components are assembled as described below. The final volume should be 20 µL.
1. Thaw all reagents on ice.
2. Assemble reaction mix into 20 µL volume in a thin walled 0.2 mL PCR tubes as follow:
    8uL ddH2O
    1µL DNA Template
    0.5uL Primer F
    0.5uL Primer R
    10uL 2x Taq mixture
3. Prepare negative control reaction without template DNA. Prepare positive control reaction with template of known size and appropriate primers.
4. Gently mix by tapping tube. Briefly centrifuge to settle tube contents.
5. Put the tube into a PCR instrument.
6. Set up the following PCR program: initial denaturing at 94°C for 4 min, followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min, plus a final extension at 72°C for 10 min.
7. Check the PCR product by agarose gel electrophoresis.

## Gel Electrophoresis

---

1. Prepare 100mL 1xTAE buffer with 1g agarose, and boil it three times, shake completely, and waiting for cool.
2. Pour the agarose gel into gel tray, assemble gel pouring apparatus by inserting gate into slots.
3. Allow agarose to cool, place the gel in the apparatus rig with the wells facing the negative end (black-colored).
4. Fill the rig with 1x TAE buffer.
5. Load 8μL of DNA maker into lane.
6. Mix 1μL of 10x loading buffer with 5μL DNA sample, load them into lane.
7. Run at 100V for 30 min.
8. Use the Gel imaging system to check the gel and take a picture.
9. Deal with the gel carefully as medical waste.

## Preparation of mutant Engineering *E.coli* by homologous recombination

---

1. Design knock-out primers according to the sequence of knock-out target gene, *lacZ*, and replace kanamycin gene for screening. The primers were designed as follows.
    Forward primer: 5’ATGACCATGATTACGGATTCACTGGCCGTCGTG TAGGCTGGAGCTGCTTC3’
    Reverse primer: 5’TTATTTTTGACACCAGACCAACTGGTAATGATGG GAATTAGCCATGGTCC3’
2. The PCR system was prepared, and the pET-28a plasmid was used as the template to amplify kanamycin gene with the homologous arm primers.
3. After the PCR reaction, the gene fragment with a size of about 1.6 Kb was identified by electrophoresis.
4. The PCR product was purified and obtained the homologous arm fragment containing kanamycin gene.
5. The homologous arm linear fragment obtained above was mixed with competent cells of *E. coli* BL21 (DE3), transfected by heat shock method at 42°C, and then the bacterial solution was coated on LB plates containing kanamycin resistance to obtain BL21 ∆ *lacZ* mutant engineering strain.
6. To further verify the kanamycin resistant BL21 mutant strain with *lacZ* gene deletion, colony PCR method was used to confirm it. The *lacZ* primers are as follows: *lacZ*-F: 5’ ATGACCATGATTACGGATTCACTGG3’ and *lacZ*-R: 5’ TTATTTTTGACACCAGACCAACTGG3’. The PCR protocol refers to the above.

## Preparation of *E. coli* BL21 Δ*LacZ* cell lysate extract

---

1. Choose the single colonies of the engineered bacteria constructed above and inoculate them in 5 ml LB medium, and culture them overnight at 37°C at 220 rpm in a shaker.
2. The next day, the medium above was transferred to 300 mL of 2 × YTPG medium, and cultured at 37°C on a shaker at 220 rpm.
3. When the OD600 value of the bacterial concentration grows at the later logarithmic period, the bacteria are collected. Centrifuge 10000g for 1 min and remove the supernatant.
4. Suspend the bacteria with the precooled S30 buffer, mix well with a shaker, and mix at 4°C and 8000g for 7 min and remove the supernatant. This step is repeated 3 times, and all traces of the supernatant is finally discarded.
5. Add 1 mL precooled S30 buffer to every 1g of bacteria and mix well.
6. Cells were crushed with a ultrasonic cell breaker, turn on for 2S, turn off for 2S, the total time is 15m, and the temperature alarm is set at 40°C.
7. Centrifuge the tube at 4°C, 12000g for 20 min and take the supernatant into a new tube, freeze it in liquid nitrogen, and store it in the refrigerator at -80°C. Ready for use as the lysate extract.
Note: 2 × YTPG medium:22 mM potassium dihydrogen phosphate, 40 Mm dipotassium hydrogen phosphate, 100 mM glucose, 16 g/L tryptone, 10 g/L yeast extract, 5 g/L sodium chloride. S30 Buffer: 10 mM Tris-acetate (pH8.2), 14 mM magnesium acetate, 60 mM potassium glutamate, 2 mM DTT.

## Construction of cell-free system

---

1. Prepare 11 mixtures in advance: 9 mM magnesium acetate, 90 mM potassium glutamate, 80 mM ammonium acetate, 57 mM HEPES-KOH, 0.171 mg/mL tRNA, 0.034 mg/mL folic acid, 2 mM dithiothreitol, 1 mM putrescine, 1.5 mM spermidine, 4 mM oxalic acid, 33 mM sodium pyruvate.
2. Prepare cell-free reaction system (15 µL): 6 µL 11 mixtures, 1.2 mM ATP, 0.86 mM GTP, CTP and UTP, 5% (V/V) PEG-8000, 0.1 mM phosphoenolpyruvate （PEP）, 0.27 U/µL RNase inhibitor, 2 mM 20 kinds of amino acids, 25% (V/V) *E. coli* BL21 Δ Lac Z cell lysate extract, 5% (V/V) 20 mg/mL X-gal chromogenic substrate.
3. Add 5 nM pET-28a-miR-34a-5p-*lacZ* toehold switch plasmid.
4. Incubate at 37°C for 1 h, and record the color change of the solution.
Note: use colored protein as indicator, add corresponding toehold switch plasmid, and do not add X-gal substrate.

## Preparation of Filter-Paper-Based Sensing Strips

---

1. Put the Whatmann filter paper strips (0.6 × 4 cm) into the culture dish, add 5% bovine serum protein solution to completely cover the paper, and block it overnight at 4°C.
2. The next day, discard the 5% bovine serum protein solution in the culture dish, add ddH2O to moisten the filter paper, place the culture dish in decoloration shaker, incubate for 5 min, and discard ddH2O.
3. Repeat step 2 and wash the filter paper 5 times.
4. Open the lid of the culture dish and place it in the electric constant temperature drying oven. Dry the filter paper for use.
5. Spot a volume of 7 μL of *E. coli* BL21 Δ *LacZ* cell lysate extract on Whatmann filter paper strips at spots pre-marked with a pencil.
6. Put it in an ultra-low temperature refrigerator at - 80°C for 6 h.
7. After precooling in the freeze dryer, the paper pieces are sealed with cling film, and the cling film is perforated for ventilation to freeze dry overnight.
8. Stored the filter paper sensing strips at 4°C until used.

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{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}**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.**

##

{% endblock %}
{% block page\_content %}

## Index

* [#### Overview](https://2022.igem.wiki/icjfls/results#overview)

* [#### 1. Construction and identification of positive controls for toehold switch plasmids](https://2022.igem.wiki/icjfls/results#construction)

* [#### 2. Construction and identification of toehold switch plasmids](https://2022.igem.wiki/icjfls/results#construction2)

* [#### 3. Expression of reporter proteins in BL21 strain with toehold switch plasmids and miRNAs transfection](https://2022.igem.wiki/icjfls/results#expression)

* [#### 4. Optimization of culture conditions of BL21 strains with toehold switch plasmids and miRNA transfection](https://2022.igem.wiki/icjfls/results#optimization)

* [#### 5. Preparation of cell free system and optimization of reaction conditions](https://2022.igem.wiki/icjfls/results#prepation1)

* [#### 6. Preparation of paper strip based on the cell free system and the reporter gene expression on it](https://2022.igem.wiki/icjfls/results#preparation2)

* [#### 7. Construction and identification of pET-28a-toehold switch-LacZ plasmid](https://2022.igem.wiki/icjfls/results#construction3)

* [#### 8. Obtaining Kanamycin resistant BL21 strain with LacZ gene deletion by homologous recombination](https://2022.igem.wiki/icjfls/results#obtaining)

* [#### 9. Preparation of paper strip biosensor with enzyme reaction system for β-galactosidase](https://2022.igem.wiki/icjfls/results#preparation3)

* [#### Conclusion](https://2022.igem.wiki/icjfls/results#conclusion)

* [#### Future plan and consideration](https://2022.igem.wiki/icjfls/results#future)

##

## 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.

[![](https://static.igem.wiki/teams/4167/wiki/fig-23-up-moving.jpg)](https://2022.igem.wiki/icjfls/results#index)
**Return to index**

##

## 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 *Eco*RⅠ and *Hin*d 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.

![](https://static.igem.wiki/teams/4167/wiki/fig-1-the-identification-of-pet-28a-amilcp-plasmid-by-restriction-endonuclease-digestion-and-pcr-assay.jpg)

**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 *Eco*RⅠ and *Hin*d Ⅲ restriction endonuclease, 3: The *amilCP* gene amplified by PCR method.

![](https://static.igem.wiki/teams/4167/wiki/fig-2-the-identification-of-pet-28a-mrfp-plasmid-by-restriction-endonuclease-digestion-and-pcr-assay.jpg)

**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 *Eco*RⅠ and *Hin*d Ⅲ restriction endonuclease, 3: The *mRFP* gene amplified by PCR method.

![](https://static.igem.wiki/teams/4167/wiki/fig-3-the-identification-of-pet-28a-amilgfp-plasmid-by-restriction-endonuclease-digestion-and-pcr-assay.jpg)

**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 *Eco*RⅠ and *Hin*d 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.

![](https://static.igem.wiki/teams/4167/wiki/fig-4-expression-of-chromoproteins-in-bl21-bacteria-transfected-with-pet-28a-amilcp-pet-28a-mrfp-and-pet-28a-amilgfp-plasmid-respectively.jpg)

**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.

[![](https://static.igem.wiki/teams/4167/wiki/fig-23-up-moving.jpg)](https://2022.igem.wiki/icjfls/results#index)
**Return to index**

##

##

## 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 *Eco*R I-*Hin*d 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 (*Eco*R I and *Hin*d 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).

![](https://static.igem.wiki/teams/4167/wiki/fig-5-identification-of-pet-28a-toehold-switch-amilcp-pet-28a-toehold-switch-mrfp-and-pet-28a-toehold-switch-amilgfp.jpg)

**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 by*Eco*R I and *Hin*d Ⅲ restriction endonucleases, 2: pET-28a-toehold switch-*amilCP* plasmid, 3: pET-28a-toehold switch-*mRFP* plasmid digested by *Eco*RⅠ and *Hin*d Ⅲ restriction endonucleases, 4: pET-28a-toehold switch-*mRFP* plasmid, 5: pET-28a-toehold switch-*amilGFP* plasmid digested by *Eco*RⅠ and *Hin*d 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).

![](https://static.igem.wiki/teams/4167/wiki/fig-6-expression-of-amilcp-protein-in-bl21-strain.jpg)

**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).

![](https://static.igem.wiki/teams/4167/wiki/fig-7-expression-of-mrfp-protein-in-bl21-strain.jpg)

**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).

![](https://static.igem.wiki/teams/4167/wiki/fig-8-expression-of-amilgfp-protein-in-bl21-strain.jpg)

**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).

![](https://static.igem.wiki/teams/4167/wiki/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.jpg)

**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.

![](https://static.igem.wiki/teams/4167/wiki/fig-10-the-effect-of-temperature-on-the-expression-of-reporter-proteins-in-bl21-strains-with-toehold-switch-plasmids-transfected-with-their-corresponding-mirnas.jpg)

**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℃.

![](https://static.igem.wiki/teams/4167/wiki/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.jpg)

**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.

![](https://static.igem.wiki/teams/4167/wiki/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.jpg)

**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.

Temperature is a primary factor affecting protein expression in vitro, and the optimal temperature for different proteins to express in vitro is different. The optimization results showed that cell free expression system can express reporter protein in a wide range of reaction temperatures, ranging from 25 °C to 42 °C, but the best temperature is 30 °C, as shown in Fig.13. The rotation rate has no effect on it during reaction, so 0 rpm is chosen considering its cost (Fig.14). When the reaction lasts for 1h, the reaction is almost over, so 1h is chosen as the best reaction time (Fig.15).

![](https://static.igem.wiki/teams/4167/wiki/fig-13-the-optimization-of-temperature-for-the-reporter-protein-expression-in-cell-free-system.jpg)

**Fig.13 The optimization of temperature for the reporter protein expression in cell free system.**

![](https://static.igem.wiki/teams/4167/wiki/fig-14-the-optimization-of-rotation-rate-for-the-reporter-protein-expression-in-cell-free-system.jpg)

**Fig.14 The optimization of rotation rate for the reporter protein expression in cell free system.**

![](https://static.igem.wiki/teams/4167/wiki/fig-15-the-optimization-of-reaction-time-for-the-reporter-protein-expression-in-cell-free-system.jpg)

**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.

![](https://static.igem.wiki/teams/4167/wiki/fig-16-reporter-gene-expression-triggered-by-different-concentration-of-mirna-on-the-paper-strip-sensor-based-on-the-cell-free-reaction-system.jpg)

**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 *Eco*RⅠand*Hin*d 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)

![](https://static.igem.wiki/teams/4167/wiki/fig-17-identification-of-pet-28a-toehold-switch-lacz-plasmid.jpg)

**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 *Eco*RⅠ and *Hin*d 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 (BL21D*LacZ*). 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 (BL21D*LacZ*). 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 (BL21D*LacZ*) was obtained successfully.

![](https://static.igem.wiki/teams/4167/wiki/fig-18-the-pcr-result-of-kanamycin-gene-using-pet-28a-vector-as-a-template.jpg)

**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.

![](https://static.igem.wiki/teams/4167/wiki/fig-19-the-pcr-results-of-kanamycin-and-lacz-genes-in-kanamycin-resistant-bl21-lacz-strain.jpg)

**Fig.19 The PCR results of *Kanamycin* and *LacZ* genes in kanamycin resistant BL21*****D******LacZ*** **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 BL21Δ*LacZ* 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).

![](https://static.igem.wiki/teams/4167/wiki/fig-20-the-optimization-of-temperature-for-reactions-catalyzed-by-galactosidase-in-cell-free-system.jpg)

**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.

![](https://static.igem.wiki/teams/4167/wiki/fig-21-the-optimization-of-reaction-time-for-galactosidase-catalyzing-reaction-in-cell-free-system.jpg)

**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.

![](https://static.igem.wiki/teams/4167/wiki/fig-22-the-optimization-of-mir-34a-5p-concentration-for-galactosidase-catalyzing-reaction-after-1h.jpg)

**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

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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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{% endblock %}