iGEM 2023 — Team hsasnu

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% 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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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}**We take a part as an example to demonstrate our engineering success for showing our effort.** {% endblock %}
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#### Index

* [##### Background](https://2023.igem.wiki/hsasnu/engineering#heading-1)
* [##### Engineering problem](https://2023.igem.wiki/hsasnu/engineering#heading-2)* [##### Engineering process](https://2023.igem.wiki/hsasnu/engineering#heading-3)
    + [###### Design Stage](https://2023.igem.wiki/hsasnu/engineering#heading-4)
    + [###### Build Stage](https://2023.igem.wiki/hsasnu/engineering#heading-5)
    + [###### Test Stage](https://2023.igem.wiki/hsasnu/engineering#heading-6)
    + [###### Learn Stage](https://2023.igem.wiki/hsasnu/engineering#heading-7)
    + [###### Re-Design](https://2023.igem.wiki/hsasnu/engineering#heading-8)
    + [###### Re-Build](https://2023.igem.wiki/hsasnu/engineering#heading-9)
    + [###### Re-Test](https://2023.igem.wiki/hsasnu/engineering#heading-10)
    + [###### Re-Learn](https://2023.igem.wiki/hsasnu/engineering#heading-11)
  * [##### Standard part construction](https://2023.igem.wiki/hsasnu/engineering#heading-12)
  * [##### Summary](https://2023.igem.wiki/hsasnu/engineering#heading-13)

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| --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4677/wiki/e.png) |  | **In this competition season, we constructed some parts which are involved in our project. Here, we use the new part BBa\_K4677003 (Part: [BBa K4677003 - parts.igem.org](http://parts.igem.org/Part%3ABBa_K4677003)) as an example to demonstrate our engineering success, following the engineering design cycle to show our effort.** |

## Background

---

The TMAO reductase pathway involves some proteins in *E.coli*. To understand this pathway of TorCAD expression regulated by TorR dependently on TMAO, we drew a schematic diagram to show this process (Fig.1). In the presence of TMAO, TorT-TorS form an asymmetric 2:2 complex that binds TMAO with negative cooperatives to form a symmetric active kinase. TMAO-stimulated TorS phosphorylates the response regulator TorR; phosphorylated TorR activates transcription from the TorCAD operon to express TorC, TorA and TorD in which *TorA* encodes a structural reductase, catalyzing TMAO reduction to allow anaerobic growth on nonfermentable sources.

![](https://static.igem.wiki/teams/4677/wiki/e-1.png)

**Fig.1 The TMAO reductase pathway to show the TorCAD expression regulated by TorR dependently on TMAO.**

To deeply understand the structure of TorT-TorS complex and TMAO binding site, Fig.2 was cited from reference [1].

![](https://static.igem.wiki/teams/4677/wiki/e-2.jpg)

**Fig.2 Structure of the TorT-TorS Complex and TMAO Binding Site.**
(a): Ribbon diagram of the complex viewed down dyad axis into the membrane. (b) : Ribbon diagrams of the TMAO binding sites (Asp42, Tyr44, Trp45, Tyr71, Trp140, and Tyr252) of TorT.

[![](https://static.igem.wiki/teams/4677/wiki/return-to-index.jpg)](https://2023.igem.wiki/hsasnu/engineering#heading-1)
**Return to index**

## Engineering problem

---

To establish the filter paper sensor to detect TMAO, we need to construct some recombinant plasmids containing *TorT* and *TorS* genes. As we know from Fig.1, TorT-TorS form a complex heterotetramer, in order to reduce the number of constructed plasmids, we planned that *TorT* and *TorS* genes are recombinant together for construction. But how to design it?

## Engineering process

---

|  |  |  |  |
| --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4677/wiki/e-x.png) |  | Design Stage  ---   Since *TorR-LacZ* were constructed into pET-28a (kan+) plasmid, and co-transformation of *TorR-LacZ* with TorT-TorS are required for LacZ expression in cells, the *TorT-TorS* genes were designed to insert into pET-22b (Amp+) for different antibiotic screening. *TorT* gene contains 1029 bp, and *TorS* gene contains 2715 bp which are arranged in the same chromosome with an opposite transcriptional direction (Fig.3). |

![](https://static.igem.wiki/teams/4677/wiki/e-3.png)

**Fig.3 The structure of *TorT*, *TorS* and *TorR* gene with TorCAD promoter, indicating the opposite arrangement of *TorT* and *TorS* in the same chromosome.**

To simplify the construction of the two genes, *TorT* and *TorS* are designed to recombine together to form a fusion gene transcribed by a same promoter of the vector. After transcription and translation, the fusion protein would be digested to separate each other. With this consideration, the fusion gene *TorT* and *TorS* are designed to insert into pET-22b vector (Fig.4).

![](https://static.igem.wiki/teams/4677/wiki/e-4.jpg)

**Fig.4 *TorT* and *TorS* genes were designed to construct into pET-22b vector, forming a fusion gene.**

|  |  |  |  |
| --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4677/wiki/e-y.png) |  | Build Stage  ---   *TorT* and *TorS* fusion gene was synthesized and flanked with *Bam*H I and *Hin*d III sites. After PCR amplification and restriction endonuclease digestion, *TorT* and *TorS* fusion gene was successfully inserted into pET-22b vector, constructing pET-22b-*TorT-TorS*. For identification, *TorT-TorS* was amplified by PCR method and digestion the recombinant plasmid using *Bam*H I and *Hin*d III. The result was shown in Fig.5. |

![](https://static.igem.wiki/teams/4677/wiki/e-5.jpg)

**Fig.5 pET-22b-*TorT-TorS* was identified using PCR method and restriction endonuclease digestion by *Bam*H I and *Hin*d III.**
 M: Marker; 1: Plasmid; 2: PCR result; 3: Digestion result.

|  |  |  |  |
| --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4677/wiki/e-z.png) |  | Test Stage  ---   For testing whether the pET-22b-*TorT-TorS* express TorT and TorS proteins or not, it was transformed to BL21 strain, after culturing about 20 h with IPTG induction using fresh LB medium added with Ampicillin, Cells were collected by centrifugation. Soluble cytoplasmic proteins were extracted after lysis and purified for identification using 6x His tag. Then SDS-PAGE electrophoresis was performed. However, the expected bands (TorT is about 40 kDa and TorS is about 110 kDa) were not observed, whereas there was a overexpressed protein band which molecule size is about 150 kDa (Fig.6). |

![](https://static.igem.wiki/teams/4677/wiki/e-6.jpg)

**Fig.6 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 overexpressed protein（about 150 kDa）in elution buffer.

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| --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4677/wiki/e-z1.png) |  | Learn Stage  ---   From the SDS-PAGE result, we figured out that the pET-22b-*TorT-TorS* could express TorT-TorS fusion protein. However it could not be digested to separate in cells, indicating a failed construction. So, we need to reconstruct pET-22b-*TorT-TorS* plasmid. |

[![](https://static.igem.wiki/teams/4677/wiki/return-to-index.jpg)](https://2023.igem.wiki/hsasnu/engineering#heading-1)
**Return to index**

|  |  |  |  |
| --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4677/wiki/e-x.png) |  | Re-Design  ---   To get the separate TorT and TorS proteins, with more consideration, *TorT* and *TorS* genes are recombined using a RBS sequence followed by extra 6 bases, making them transcribed together (controlled by the same promoter), but translated separately (Fig.7). |

![](https://static.igem.wiki/teams/4677/wiki/e-7.png)

**Fig.7 *TorT* and *TorS* genes are re-designed to combine together using RBS sequence followed by extra 6 bases, making them translated separately.**

|  |  |  |  |
| --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4677/wiki/e-y.png) |  | Re-Build  ---   *TorT* and *TorS* were designed to combine together using RBS (B0034) followed by extra 6 bases, synthesized and also flanked with *Bam*H I and *Hin*d III sites. Then they were inserted into pET-22b vector, constructing pET-22b-*TorT-TorS* again. The identification was performed by PCR and digestion with *Bam*H I and *Hin*d III. The result was shown in Fig.8. |

![](https://static.igem.wiki/teams/4677/wiki/e-8.jpg)

**Fig.8 Identification of pET-22b-TorT-TorS plasmid.**

M: Marker; 1: The plasmid of pET-22b-*TorT-TorS*; 2: The pET-22b-*TorT-TorS* plasmid digested by *Bam*H I and *Hin*d Ⅲ restriction endonuclease; 3: The *TorT-TorS* genes amplified with PCR method.

|  |  |  |  |
| --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4677/wiki/e-z.png) |  | Re-Test  ---   To test the new plasmid whether express the TorT and TorS proteins separately, same procedure was performed. Then SDS-PAGE electrophoresis was also conducted. As expected this time, two overexpressed bands (TorT 40 kDa and TorS 110 kDa) were observed in Fig.9. |

![](https://static.igem.wiki/teams/4677/wiki/e-9.jpg)

**Fig.9 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.

|  |  |  |  |
| --- | --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/4677/wiki/e-z1.png) |  | Re-Learn  ---   From the result of re-test, we know that the new recombinant plasmid pET-22b-*TorT-TorS* was constructed successfully. So, it can be used for detection TorCAD promoter activity regulated by TorR dependently on TMAO concentration. To contribute to the iGEM community in future, we built a standard part in the following. |

[![](https://static.igem.wiki/teams/4677/wiki/return-to-index.jpg)](https://2023.igem.wiki/hsasnu/engineering#heading-1)
**Return to index**

## Construction of the standard part pSB1C3-*TorT-TorS* (K4677003)

---

In order to construct the standard part pSB1C3-*TorT-TorS* plasmid, *TorT-TorS* sequence was tested to see if there are *Eco*R I and *Pst* I sites. The testing result was shown in Fig.10.

![](https://static.igem.wiki/teams/4677/wiki/e-10.jpg)

**Fig.10 The map of *TorT-TorS* sequence described by SnapGene Viewer, showing the restriction enzyme information (no *Eco*R I and *Pst* I sites).**

After testing the restriction enzyme information of *TorT-TorS* gene using SnapGene software, it was inserted into the pSB1C3 plasmid to construct the standard part (BioBrick) pSB1C3-*TorT-TorS* with PCR method. Then it was identified as follows (Fig.11):

![](https://static.igem.wiki/teams/4677/wiki/e-11.jpg)

**Fig.11 Identification of standard part pSB1C3-*TorT-TorS* using PCR method and digestion with *Eco*R I and *Pst* I.**
M: Marker; 1: Plasmid; 2: PCR result; 3: Digestion result.

[![](https://static.igem.wiki/teams/4677/wiki/return-to-index.jpg)](https://2023.igem.wiki/hsasnu/engineering#heading-1)
**Return to index**

## Summary

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This part (K4677003) was aimed to construct for co-transformation with pET-28a-*TorR-LacZ* plasmid, detecting the activity of TorCAD promoter regulated by TorR in the presence of TMAO. *LacZ* serves as a reporter gene which encodes β-galactosidase to catalyze the substrate X-gal, generating blue product (Fig.12), which could be quantified. For more information, please refer to our another part K4677005 (Part: [BBa K4677005 - parts.igem.org](http://parts.igem.org/Part%3ABBa_K4677005)) or our result ([Results | HSASNU - iGEM 2023](https://2023.igem.wiki/hsasnu/results)).

![](https://static.igem.wiki/teams/4677/wiki/e-12.png)

**Fig.12 The blue clones were observed on the agar plate when TorR-TorCAD promoter-*LacZ* and *TorT-TorS* genes were co-transformed into BL21D*LacZ* in the presence of TMAO and X-gal.**
 A: Negative control without TMAO; B: experiment group with TMAO (100 µM) and X-gal (40 µg/mL).

[![](https://static.igem.wiki/teams/4677/wiki/return-to-index.jpg)](https://2023.igem.wiki/hsasnu/engineering#heading-1)
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##### References

[1] Moore JO, Hendrickson WA. An asymmetry-to-symmetry switch in signal transmission by the histidine kinase receptor for TMAO. Structure. 2012; 20(4): 729-741.doi:10.1016/j.str.2012.02.021

[2] Tang WH, Wang Z, Levison BS, Koeth RA, Britt EB, Fu X, et al. Intestinal microbial metabolism of phosphatidylcholine and cardiovascular risk. N Engl J Med 2013; 368(17):1575-1584.

[3] Senthong V, Wang Z, Fan Y, Wu Y, Hazen SL, Tang WH. Trimethylamine N-Oxide and Mortality Risk in Patients With Peripheral Artery Disease. J Am Heart Assoc 2016; 5(10)

[4] Senthong V, Wang Z, Li XS, Fan Y, Wu Y, Tang WH, et al. Intestinal Microbiota-Generated Metabolite Trimethylamine-N-Oxide and 5-Year Mortality Risk in Stable Coronary Artery Disease: The Contributory Role of Intestinal Microbiota in a COURAGE-Like Patient Cohort. J Am Heart Assoc 2016; 5(6)

[5] Zhu Y, Li Q, Jiang H. Gut microbiota in atherosclerosis: focus on trimethylamine N-oxide. APMIS. 2020;128(5):353-366. doi:10.1111/apm.13038

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{% block title %}Experiments{% endblock %}
{% block lead %} **We list our experimental protocols in our iGEM project.**{% endblock %}
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## Overview

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In our project, we established an early warning system for atherosclerosis (AS) by detecting trimethylamine N-oxide (TMAO) and microRNA (miRNA) in urine. We have developed a test strip sensor based on toe switches and a cell-free expression system to detect TMAO and miRNA, which helps to provide early warning of AS at home. The experimental protocol is shown below.

## 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:
        8 µL ddH2O
        1 µL DNA Template
        0.5 µL Primer F
        0.5 µL Primer R
        10 µL 2 x 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.

## Agarose electrophoresis

---

1). Prepare 100 mL 1 x TAE buffer with 1 g 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 1 x TAE buffer.
5). Load 8 μL of DNA maker into lane.
6). Mix 1 μL of 10 x loading buffer with 5 μL DNA sample, load them into lane.
7). Run at 100 V 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.

## 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 3 mL of LB broth solution. Incubate it at 37°C overnight (more than 16 h) with shaking at 220 rpm.
3). Transfer 250 μL of the saturated overnight solution to a sterile 50 mL polypropylene tube containing 25 mL 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 value 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 8,000 rpm for 1 min at 4°C, discard the supernatant.
5). Re-suspend the *E. coli* in 2.5 mL of ice-cold TSS solution with gentle swirling. Incubate on ice for 10 min. Aliquot in 50 μL 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 8,000) 10% 1.0 g, DMSO (vol/vol) 5% 500 μL, and MgCl2 (pH 6.5) 50 mM 500 μL.

## Plasmid transforming and pick monoclone

---

1). Thaw TSS cells on ice.
2). Add 5 µL 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 0.8 mL LB medium into the cells.
6). Shake and incubate at 37 °C for 60 min at 120 rpm.
7). Take out 100 μL medium and spread them on the appropriate agar plates with certain antibiotic.
8). Incubate the plates at 37 °C overnight.
9). Pick monoclone 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 1,500 µL solution of cultivated bacteria to a 2 mL micro-centrifuge tube. The tube was centrifuged at 12,000 rpm 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 250 µL 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 250 µL 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 350 µL Solution III buffer, mix gently and fully upside down 6-8 times, centrifuge the tubes at 12,000 rpm for 10 min at room temperature.
6). Put a HiBind DNA Mini Column into a 2 mL Collection Tube.
7). The supernatant in step 4 is transferred to the Mini Column, and centrifuged at 12,000 rpm for 1 min, the filtrate is discarded.
8). Put the Column back into the Collection Tube, add 500 µL HBC Buffer, centrifuge at 12,000 rpm for 1 min, the filtrate is discarded.
9). Put the Column back into the Collection Tube, add 700 µL Washing buffer, centrifuge at 12,000 rpm 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 1.5 mL micro-centrifuge tube and leave it for 2 min at room temperature. Then add 50 µL ddH2O to the center of the Column matrix, and leave it at room temperature for 1 min. Centrifuge at 12,000 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 20 µL.
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
    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.

## Preparation of mutant *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’ATGACCATGATTACGGATTCACTGGCCGTCGTGTAGGCTGGAGCTGCTTC3’
    Reverse primer: 5’TTATTTTTGACACCAGACCAACTGGTAATGATGGGAATTAGCCATGGTCC3’
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’
    *lacZ*-R: 5’ TTATTTTTGACACCAGACCAACTGG3’.
The PCR protocol refers to the above.

## Purification of His/S-tagged protein

---

All the procedures were performed at 4 °C or on ice unless otherwise specified.
1). 10 mL BL21 cells transformed with a pET-28a-*TorR-lacZ* or pET-28a-*TorT-TorS* were cultured at 30°C until an OD660 of approx. 0.8.
2). Add IPTG to the culture (final 0.67 mM IPTG)
3). Cultivate at 30 °C for 2 h.
4). Collect cells in 5 mL tube, centrifuge at 5,000 rpm at 15 min.
5). Suspend cells in 5 mL of TBS, transfer the content to 1.5 mL tube.
6). Collect cells at 10,000 rpm at 1 min. The supernatant was discarded.
7). Add 500 µL of ice-cold lysis buffer to the cell pellet.
8). Sonicate cell (4°C).
9). Clarify lysate by centrifugation at to 10,000 rpm at 10 min.
10). After centrifugation, save 20 µL of supernatant for SDS-PAGE sample.
11). Transfer supernatant to the 1.5mL tube containing pre-balanced resin bead gel.
12). Shake gently (4°C) for 30 min.
13). Centrifuge the tube 1,200 rpm for 30 sec. The supernatant was discarded.
14). Wash resin bead gel with washing buffer, Centrifuge the tube 1,200 rpm for 30 sec. The supernatant was discarded. Repeat it once.
15). Elute the protein 3 time with 20 µL of elution buffer. It is ready to prepare the SDS-PAGE sample.

## SDS-PAGE electrophoresis

---

1). Make up 30 mL of running gel by adding components to a clean glass in the order as follow:
    12.3 mL H2O
    7.5 mL 1.5 M Tris-HCl, pH 8.8
    0.15 mL 20% (w/v) SDS
    9.9 mL Acrylamide/Bis-acrylamide(30%/0.8% w/v)
    0.15 mL 10% (w/v) ammonium persulfate (APS)
    0.02 mL TEMED
2). Mix the ingredients mentioned above.
3). Pour the solution quickly into a gel casting form, and leave about 2 centimeters below the bottom of the comb for the stacking gel. Then layer the top of the gel with water very carefully. Wait for about 30 min for the gel to polymerize completely.
4). While waiting, mix the reagents for the stacking gel (4% Acrylamide) by adding components to a clean glass in the order as follow:
    3.075 mL H2O
    1.25 mL 0.5 M Tris-HCl, pH 6.8
    0.025 mL 20% (w/v) SDS
    0.67 mL Acrylamide/Bis-acrylamide (30%/0.8% w/v)
    0.025 mL 10% (w/v) ammonium persulfate (APS)
    0.005 ml TEMED
5). Mix the ingredients mentioned above.
6). Remove the water on the top of running gel, and pour the stacking gel on top of the running gel. Insert the combs by trying not to produce bubbles stuck underneath and allow another 0.5 - 1 h for complete polymerization.
7). Prepare samples: mix the protein 4:1 with the loading buffer. Heat the sample by Boiling for 5-10 min.
8). Run gel. Clamp the gel and fill both buffer chambers with gel running buffer. Pipet the sample and molecular weight standard marker into the gel. Attach the power leads and run the gel until the blue dye reaches the bottom.
9). Visualize the proteins using Coomassie Brilliant Blue.

## Preparation of *E. coli* BL21DLacZ 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 10,000g 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 8,000 g 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 1 g of bacteria and mix well.
6). Cells were crushed with a ultrasonic cell breaker, turn on for 2 S, turn off for 2 S, the total time is 15 min, and the temperature alarm is set at 40 °C.
7). Centrifuge the tube at 4 °C, 12,000 g 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: 12 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 toehold switch plasmid (pET-28a-miR-17-5p-*LacZ* or pET-28a-miR-146a-5p-*LacZ*) or 380 mM TMAO.
4). Incubate at 37 °C for 1 h, and record the color change of the solution.
5). Add 3 µL of reaction solution mentioned above to 297 µL of distilled water, transfer to a 96 well plate, and measure the absorbance value at OD570 nm.

## 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.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}**We established a filter paper biosensor on which 3 biomarkers (TMAO, miR-17-5p and miR-146a-5p) could be tested in a cell free system to make an early warning for AS.** {% endblock %}
{% block page\_content %}

#### Index

* [###### 1. TMAO testing system](https://2023.igem.wiki/hsasnu/results#heading-1)
* [###### 2. microRNA testing system](https://2023.igem.wiki/hsasnu/results#heading-2)
* [###### 3. Construction of filter paper biosensor](https://2023.igem.wiki/hsasnu/results#heading-3)
* [###### 4. Conclusion](https://2023.igem.wiki/hsasnu/results#heading-4)
* [###### 5. Future plan and consideration](https://2023.igem.wiki/hsasnu/results#heading-5)

![](https://static.igem.wiki/teams/4677/wiki/heji-8ge2.png)

## 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 *Bam*H I and *Hin*d 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 *Bam*H I and *Hin*d 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 *Bam*H I and *Hin*d 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.

![](https://static.igem.wiki/teams/4677/wiki/r-fig-1-identification-of-pet-28a-torr-lacz-plasmid.jpg)

**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 *Bam*H I and *Hin*d Ⅲ restriction endonuclease; 3: The *TorR-lacZ* gene amplified with PCR method.

![](https://static.igem.wiki/teams/4677/wiki/r-fig-2-identification-of-pet-28a-tort-tors-plasmid.jpg)

**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 *Bam*H I and *Hin*d Ⅲ restriction endonuclease; 3: The *TorT-TorS* gene amplified with PCR method.

[![](https://static.igem.wiki/teams/4677/wiki/return-to-index.jpg)](https://2023.igem.wiki/hsasnu/results#overview)
**Return to index**

##### **1.2 Expression and purification of *TorR*, *TorT* and *Tor*S 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.

![](https://static.igem.wiki/teams/4677/wiki/r-fig-3-the-sds-page-result-shows-expression-and-purification-of-torr-protein.jpg)

**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.

![](https://static.igem.wiki/teams/4677/wiki/r-fig-4-the-sds-page-result-shows-expression-and-purification-of-tort-and-tors-proteins.jpg)

**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.

[![](https://static.igem.wiki/teams/4677/wiki/return-to-index.jpg)](https://2023.igem.wiki/hsasnu/results#overview)
**Return to index**

##### **1.3 Construction and identification of Kanamycin resistant BL21 strain with *LacZ* gene deletion (BL21D*LacZ*)**

To express the recombinant *LacZ* gene, we constructed BL21 (DE3) mutant strain with *LacZ* gene deletion (BL21D*LacZ*). 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 (BL21D*LacZ*). 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 (BL21D*LacZ*) was constructed successfully.

![](https://static.igem.wiki/teams/4677/wiki/r-fig-5-the-pcr-result-of-kanamycin-gene-using-pet-28a-vector-as-a-template-n.jpg)

**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.

![](https://static.igem.wiki/teams/4677/wiki/r-fig-6-the-pcr-results-of-kanamycin-and-lacz-genes-in-kanamycin-resistant-bl21dlacz-strain-n.jpg)

**Fig.6 The PCR results of *Kanamycin* and *LacZ* genes in kanamycin resistant BL21D*LacZ* strain.**
M: Marker； 1: *Kanamycin* gene. 2. No band of *LacZ* gene amplified with PCR method.

[![](https://static.igem.wiki/teams/4677/wiki/return-to-index.jpg)](https://2023.igem.wiki/hsasnu/results#overview)
**Return to index**

##### **1.4 Induced expression of LacZ triggered by TMAO in BL21D*LacZ* 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 BL21D*LacZ* strain. Since there is no TMAO in BL21D*LacZ* 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, BL21D*LacZ* 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.

![](https://static.igem.wiki/teams/4677/wiki/r-fig-7-the-induced-expression-of-lacz-galactosidase-in-bl21dlacz-strain-co-transformed-with-pet-28a-torr-lacz-and-pet-22b-tort-tors-plasmids.jpg)

**Fig.7 The induced expression of LacZ in BL21D*LacZ* 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.

[![](https://static.igem.wiki/teams/4677/wiki/return-to-index.jpg)](https://2023.igem.wiki/hsasnu/results#overview)
**Return to index**

##### **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 BL21D*LacZ* 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).

![](https://static.igem.wiki/teams/4677/wiki/r-fig-8-the-optimization-of-reactional-conditions-catalized-by-galactosidase-in-cell-free-system-to-increase-the-sensitivity-of-tmao-detection.jpg)

**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.

![](https://static.igem.wiki/teams/4677/wiki/r-fig-9-the-quantitative-analysis-of-conditional-optimization.jpg)

**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.

[![](https://static.igem.wiki/teams/4677/wiki/return-to-index.jpg)](https://2023.igem.wiki/hsasnu/results#overview)
**Return to index**

## 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 (*Bam*H I and *Hin*d 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).

![](https://static.igem.wiki/teams/4677/wiki/r-fig-10-identification-of-pet-28a-mir-17-5p-toehold-switch-lacz-plasmid.jpg)

**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 *Bam*H I and  *Hin*d III restriction endonuclease; 3: The inserted fragment containing *LacZ* gene amplified with PCR method.

![](https://static.igem.wiki/teams/4677/wiki/r-fig-11-identification-of-pet-28a-mir-146a-5p-toehold-switch-lacz-plasmid.jpg)

**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 *Bam*H Ⅰ and *Hin*d Ⅲ restriction endonuclease; 3: The inserted fragment containing *LacZ* gene amplified with PCR method.

##### **2.2 Induced expression of LacZ by microRNAs in BL21D*LacZ* 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 BL21D*LacZ* 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 BL21D*LacZ* 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.

![](https://static.igem.wiki/teams/4677/wiki/r-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.jpg)

**Fig.12 Induced expression of *LacZ* gene by miR-17-5p in BL21D*LacZ* strain transformed with pET-28a-miR-17-5p toehold switch-*lacZ* plasmid.**
(a) Without miR-17-5p, BL21D*LacZ* 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, BL21D*LacZ* 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.

![](https://static.igem.wiki/teams/4677/wiki/r-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.jpg)

**Fig.13 Induced expression of *LacZ* gene by miR-146a-5p in BL21D*LacZ* strain transformed with pET-28a-miR-146a-5p toehold switch-*LacZ* plasmid.**
(a) Without miR-146a-5p, BL21D*LacZ* 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, BL21D*LacZ* 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.

[![](https://static.igem.wiki/teams/4677/wiki/return-to-index.jpg)](https://2023.igem.wiki/hsasnu/results#overview)
**Return to index**

##### **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 BL21D*LacZ* 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.

![](https://static.igem.wiki/teams/4677/wiki/r-fig-14-the-optimization-of-reactional-conditions-catalized-by-galactosidase-in-cell-free-system-to-increase-the-sensitivity-of-mir-17-5p-detection.jpg)

**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..

![](https://static.igem.wiki/teams/4677/wiki/r-fig-15-the-quantitative-analysis-of-conditional-optimization-for-detection-of-mir-17-5p-using-cell-free-systerm.jpg)

**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.

![](https://static.igem.wiki/teams/4677/wiki/r-fig-16-the-optimization-of-reactional-conditions-catalized-by-galactosidase-in-cell-free-system-to-increase-the-sensitivity-of-mir-146a-5p-detection.jpg)

**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..

![](https://static.igem.wiki/teams/4677/wiki/r-fig-17-the-quantitative-analysis-of-conditional-optimization-for-detection-of-mir-146a-5p-using-cell-free-systerm.jpg)

**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.

[![](https://static.igem.wiki/teams/4677/wiki/return-to-index.jpg)](https://2023.igem.wiki/hsasnu/results#overview)
**Return to index**

##

## 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 BL21D*lacZ* strains**

Three kinds of cell free systems were required to prepare. All of them were extracted from BL21D*lacZ* strains co-transformed with pET-28a-*TorR-**lacZ* and pET-22b-*TorT-Tor*S 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](https://2023.igem.wiki/hsasnu/experiments) 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.

![](https://static.igem.wiki/teams/4677/wiki/r-fig-18-the-optimization-result-for-detecting-100-m-tmao-in-cell-free-system-on-the-filter-paper-strip.jpg)

**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.

![](https://static.igem.wiki/teams/4677/wiki/r-fig-19-the-optimization-result-for-detecting-500-pm-mir-17-5p-in-cell-free-system-on-the-filter-paper-strip.jpg)

**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.

![](https://static.igem.wiki/teams/4677/wiki/r-fig-20-the-optimization-result-for-detecting-500-pm-mir-146a-5p-in-cell-free-system-on-the-filter-paper-strip.jpg)

**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.

![](https://static.igem.wiki/teams/4677/wiki/r-fig-21-detecting-tmao-mir-146a-5p-and-mir-17-5p-on-the-filter-paper-biosensor-at-the-same-time.jpg)

**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

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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

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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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