iGEM 2024 — Team icjfls

{% extends "layout.html" %}
{% 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 %}
{% block page\_content %}

## What should this page contain?

---

* 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

---

* [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)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %} **We demonstrated the engineering design cycle and the construction of a new part to show our effort for engineering success.**{% endblock %}
{% block page\_content %}

#### Index

* [###### Engineering design cycle](https://2024.igem.wiki/icjfls/engineering#heading-2)

  + [###### Design Stage](https://2024.igem.wiki/icjfls/engineering#heading-3)

  + [###### Build Stage](https://2024.igem.wiki/icjfls/engineering#heading-4)

  + [###### Test Stage](https://2024.igem.wiki/icjfls/engineering#heading-5)

  + [###### Learn Stage](https://2024.igem.wiki/icjfls/engineering#heading-6)

  + [###### Re-Design](https://2024.igem.wiki/icjfls/engineering#heading-7)

  + [###### Re-Build](https://2024.igem.wiki/icjfls/engineering#heading-8)

  + [###### Re-Test](https://2024.igem.wiki/icjfls/engineering#heading-9)

  + [###### Re-Learn](https://2024.igem.wiki/icjfls/engineering#heading-10)

* [###### Construction of a new part](https://2024.igem.wiki/icjfls/engineering#heading-11)

## Overview

---

In this section, we would like to demonstrate our engineering design cycle to show the engineering success and effort. Also, we constructed some plasmids which are involved in our project in 2024 competition season. Here, we would like to show the construction of a new part ([BBa\_K5074001)](https://parts.igem.org/Part%3ABBa_K5074001).

|  |
| --- |
| ![](https://static.igem.wiki/teams/5074/eng-cycle-1.jpg) |

## 1. The engineering design cycle

---

### 1.1 Introduction

To establish a biosensor for detecting bacteria in refrigerator, we need to construct some recombinant plasmids containing RNA aptamer toehold switches, which can recognize and bind to some bacteria to trigger the expression of reporter genes. We used 3 reporter genes *amilCP*, *mRFP* and *EGFP* to construct 3 recombinant plasmids which are used to detect 3 bacteria *Shigella*, *Escherichia coli* and *Salmonella*, respectively. Here, we took the construction of recombinant plasmid for detecting *Escherichia coli* (*E.coli*) as an example to demonstrate the engineering success.

### 1.2 Engineering cycle

|  |  |  |
| --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/5074/eng-cycle-2.jpg) | 1.2.1 Design Stage |

---

According to the aptamer sequence of *E.coli* and the construction rules of toehold switch, we designed the aptamer toehold switch with mRFP reporter gene which was aimed to detect *E.coli*. At the presence of *E.coli* or the complementary fragment of aptamer (trigger RNA), it can bind to the toehold switch and trigger the expression of reporter gene (Fig.1).

![](https://static.igem.wiki/teams/5074/eng-fig-1.jpg)

**Fig.1 The construction principle of RNA Toehold Switch using *mRFP* reporter gene.**

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/engineering#early)
**Return to index**

|  |  |  |
| --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/5074/eng-cycle-3.jpg) | 1.2.2 Build Stage |

---

The sequence of aptamer toehold switch was synthesized and inserted into pET-28a-*mRFP* plasmid using PCR method. This plasmid was named as pET-28a*-Esch-mRFP*. After the construction, it was identified using PCR amplification. The inserted fragment length was about 850 bp. The agarose electrophoresis result showed that the PCR result was consistent with the expected length (Fig.2).

![](https://static.igem.wiki/teams/5074/eng-fig-2.jpg)

**Fig.2 Electrophoresis Identification of pET-28a-*Esch-mRFP* aptamer toehold switch plasmid and PCR amplification.**
M: Marker, 1: Plasmid of pET-28a-*Esch-mRFP*, 2: The PCR result of aptamer toehold switch *mRFP*.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/engineering#early)
**Return to index**

|  |  |  |
| --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/5074/eng-cycle-4.jpg) | 1.2.3 Test Stage |

---

Then the constructed plasmid was transformed into BL21 cells, and the result showed that no red clone was observed (Fig.3A), illustrating the aptamer toehold switch plasmid was constructed successfully, which red protein didn’t express because of the translation inhibition by aptamer toehold switch.

![](https://static.igem.wiki/teams/5074/eng-fig-3.jpg)

**Fig.3 The plasmid** **pET-28a-*Esch-mRFP*** **was transformed into BL21 cells, no red clone was observed, compared to the BL21 cells transformed with pET-28a-*mRFP* plasmid.**
(A) BL21 cells transformed with pET-28a-*Esch-mRFP*plasmid; (B) BL21 cells transformed with pET-28a-*mRFP* plasmid.

To study whether the *E.coli* aptamer toehold switch work or not, the complementary fragment of RNA aptamer of *E.coli* was synthesized. Then both the complementary fragment of RNA aptamer and the plasmid pET-28a-*Esch-mRFP* were transformed into BL21 cells. The expression of reporter gene mRFP was observed (Fig.4), indicating that the aptamer toehold switch can control the expression of reporter gene, and the complementary fragment of RNA aptamer can trigger the expression of reporter gene.

![](https://static.igem.wiki/teams/5074/eng-fig-4.jpg)

**Fig.4 After transformation of both complementary fragment of RNA aptamer and the pET-28a-*Esch-mRFP* plasmid, the expression of reporter gene mRFP was observed.**

To optimize the expression of reporter gene mRFP using the complementary fragment of aptamer and cell free system which is used for making hydrogel to detect *E.coli* in fridge, we prepared the cell free system extracted from BL21 cells transformed with pET-28a-*Esch-mRFP* plasmid. Add the complementary fragment of aptamer to the cell free system to observe the reporter gene expression.

The reaction conditions for expressing reporter protein in cell free system were optimized. The conditions including temperature, concentration of fragment, and reaction time were optimized in order to obtain sensitive and fast detection effect.

The result (Fig.5) shows that cell free system can express reporter protein ranging from 4°C to 37°C, but the proper temperature varies from 25°C to 37°C for *mRFP* expression. The lowest good concentration is about 2 uM. For visible reporter protein expression, it needed 6 h at least in cell free systems.

![](https://static.igem.wiki/teams/5074/eng-fig-5.jpg)

**Fig.5 The optimization result of reporter gene expression in cell free system transformed with pET-28a-*Esch-mRFP* after adding the complementary fragment of aptamer.**
(A) temperature,1: 4℃, 2: 10℃, 3: 15℃, 4: 20℃, 5: 25℃, 6: 30℃, 7: 37℃; (B) concentration,1: 0.5 uM, 2: 1 uM, 3: 2 uM, 4: 4 uM, 5: 6 uM, 6: 8 uM, 7: 10 uM; (C) reaction time, 1: 6 h, 2: 8 h, 3: 10 h, 4: 13 h, 5: 16 h, 6: 20 h, 7: 24 h.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/engineering#early)
**Return to index**

|  |  |  |
| --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/5074/eng-cycle-5.jpg) | 1.2.4 Learn Stage |

---

We successfully constructed the plasmid pET-28a-*Esch-mRFP*, which can express reporter gene *mRFP* under the control of complementary fragment of *E.coli* aptamer. At the presence of complementary fragment of *E.coli* aptamer, *mRFP* was expressed well. But from the optimization result, we figured out that to express reporter gene *mRFP* in cell free system, 2 uM complementary fragment of aptamer is relatively high which is not sensitive, and it needs long reaction time (6 h) for detection. Both of which required to improve.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/engineering#early)
**Return to index**

|  |  |  |
| --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/5074/eng-cycle-2.jpg) | 1.2.5 Re-Design |

---

In order to improve sensitivity and reduce detection time, the report gene was replaced with *LacZ* gene which can express an enzyme, amplifying the detection result. So, the pET-28a-*Esch-lacZ* toehold switch plasmid was constructed using *lacZ* as reporter gene, used to detect *E. coli* in cell free systems. *lacZ* can express β-galactosidase, catalyzing the blue product generation using X-gal, which amplifies the detection signals (Fig.6).

![](https://static.igem.wiki/teams/5074/eng-fig-6-1.jpg)

**Fig.6 The construction principle of RNA Toehold Switch using *LacZ* reporter gene.**

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/engineering#early)
**Return to index**

|  |  |  |
| --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/5074/eng-cycle-3.jpg) | 1.2.6 Re-Builed |

---

For identification of reconstructed plasmid pET-28a-*Esch-lacZ*, the restriction endonuclease digestion and PCR assay were performed. The results showed that the fragment lengths of aptamer toehold switch with *lacZ* were consistent with the expected results, indicating that pET-28a-*Esch-LacZ* was constructed successfully (Fig.7).

![](https://static.igem.wiki/teams/5074/eng-fig-7.jpg)

**Fig.7 Identification of aptamer toehold switch plasmid pET-28a-*Esch-LacZ*.**
M: Marker, 1: The plasmid of pET-28a-*Esch-LacZ*, 2: PCR result, 3: Digestion result by *Eco*R I and Hind III restriction endonuclease.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/engineering#early)
**Return to index**

|  |  |  |
| --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/5074/eng-cycle-4.jpg) | 1.2.7 Re-Test |

---

To identify whether the *LacZ* gene of recombinant plasmid express or not，both the complementary fragment of RNA aptamer and the plasmid pET-28a-*Esch-lacZ* were transformed into BL21D*LacZ* strain (with *LacZ* deletion). After culture 12 h with fresh LB medium containing Kanamycin and Ampicillin, the LacZ protein (β-galactosidase) was purified using 6x His tag column for identification. The SDS-PAGE electrophoresis result was shown in Fig.8.

![](https://static.igem.wiki/teams/5074/eng-fig-8.jpg)

**Fig. 8 The SDS-PAGE result shows expression and purification of LacZ protein.**
M: Marker, 1: All supernatant proteins of BL21D*LacZ* strain, 2: All supernatant proteins of BL21D*LacZ* strain transformed with pET-28a-*Esch-LacZ* and the complementary fragment of *E. coli* aptamer, 3: Purified LacZ protein from the supernatant proteins of sample 2.

To test the feasibility of our constructed cell free system containing pET-28a-*Esch-LacZ* plasmid for bacteria detection. The cell free system was prepared using pET-28a-Esch-LacZ plasmid transformed BL21D*LacZ* strain. Complementary fragment and X-gal (substrate of β-galactosidase) were added to the cell free system under different conditions including temperature, concentration of complementary fragment, concentration of X-gal, and detection time. Since the maximum absorbance of the blue product is located at 615 nm, the experiment results were quantified using absorbance value, which were showed in Fig.9.

![](https://static.igem.wiki/teams/5074/eng-fig-9.jpg)

**Fig.9 The quantification results of experiment performed in cell free system containing pET-28a-*Esch-LacZ* plasmid under different conditions.**
(A) Absorbance of the end product catalyzed by β-galactosidase expressed in cell free system with 1 uM complementary fragment of aptamer and 100 ug/mL X-gal, 1 h reaction time, under different temperature; (B) Absorbance of the end product catalyzed by β-galactosidase expressed in cell free system with 100 ug/mL X-gal and different concentration of complementary fragment of aptamer,1 h reaction time, under 20℃; (C) Absorbance of the end product catalyzed by β-galactosidase expressed in cell free system with 1 uM complementary fragment of aptamer and different concentration of X-gal, 1 h reaction time, under 20℃; (D) Absorbance of the end product catalyzed by β-galactosidase expressed in cell free system with 1 uM complementary fragment of aptamer and 100 ug/mL X-gal, different reaction time, under 20℃.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/engineering#early)
**Return to index**

|  |  |  |
| --- | --- | --- |
|  | ![](https://static.igem.wiki/teams/5074/eng-cycle-5.jpg) | 1.2.8 Re-Learn |

---

The quantification results of Fig.9 indicated that the cell free system extracted from pET-28a-*Esch-LacZ* transformed BL21 cells works well. For intuitive and visible good result, the best conditions for reaction are 20℃, 0.5 uM complementary fragment of aptamer, 100 ug/mL X-gal, and 1 h reaction time, all of which are improved.

**Summary:**

Through two engineering cycles, we constructed successfully pET-28a-*Esch-LacZ* plasmid which is triggered to effectively express reporter gene by the complementary fragment of aptamer. This plasmid will be used to detect *E. coli* in the future.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/engineering#early)
**Return to index**

## 2. Construction of a new part BBa\_K5074001

---

In this competition season, we also constructed a new part ([BBa\_K5074001)](https://parts.igem.org/Part%3ABBa_K5074001). It is an *AmilCP* generator controlled by *Shigella* RNA aptamer toehold switch. At the presence of *Shigella dysenteriae,* it binds to its RNA aptamer, opening the toehold switch to trigger the expression of amilCP, which is easily detected. It is used to detect the bacteria *Shigella* in refrigerator.

This part comprises *Shigella dysenteriae* RNA aptamer, RBS (B0034), Linker and reporter gene *amilCP* (K592009). Its sequence was analyzed using SnapGene Viewer for restriction enzyme cleaving sites to construct the plasmid pET-28a-*Shig-amilCP* using the proper enzymes. The analysis result with SnapGene Viewer was shown in Fig.10.

![](https://static.igem.wiki/teams/5074/eng-fig-10-1.jpg)

**Fig.10 The endonuclease map of BBa\_K5074001 analyzed using SnapGene Viewer, showing the restriction enzyme information** (no *Eco*RI and *Hin*d III cleaving sites).

The sequence of aptamer toehold switch was synthesized and inserted into pET-28a-*amilCP* plasmid using PCR method. This plasmid was named as pET-28a-*Shig-amilCP* which was identified by PCR amplification. The inserted fragment length was about 801 bp. The agarose electrophoresis result showed that the PCR product was consistent with the expected result (Fig.11).

![](https://static.igem.wiki/teams/5074/eng-fig-11.jpg)

**Fig.11 Electrophoresis Identification of pET-28a-*Shig-amilCP* aptamer toehold switch plasmid and PCR amplification.**
M: Marker, 1: Plasmid of pET-28a-*Shig-amilCP*, 2: The PCR result of aptamer toehold switch *amilCP*.

Then the constructed plasmid was transformed into BL21 cells, and the result showed that no purple clone was observed (Fig.12A), illustrating the aptamer toehold switch plasmid was constructed successfully, which purple protein didn’t express because of the translation inhibition by aptamer toehold switch.

![](https://static.igem.wiki/teams/5074/eng-fig-12.jpg)

**Fig.12 The plasmid pET-28a-*Shig-amilCP* was transformed into BL21 cells, no purple clone was observed, compared to the BL21 cells transformed with pET-28a-*amilCP* plasmid.**
(A) BL21 cells transformed with pET-28a-*Shig-amilCP* plasmid; (B) BL21 cells transformed with pET-28a-*amilCP* plasmid.

To study whether the *Shigella* aptamer toehold switch work or not, the complementary fragment of RNA aptamer of *Shigella* was synthesized. Then both the complementary fragment of RNA aptamer and the plasmid pET-28a-*Shig-amilCP* were transformed into BL21 cells. The expression of reporter gene *amilCP* was observed (Fig.13), indicating that the aptamer toehold switch can control the expression of reporter gene, and the complementary fragment of RNA aptamer can trigger the expression of reporter gene.

![](https://static.igem.wiki/teams/5074/eng-fig-13.jpg)

**Fig.13 After transformation of both complementary fragment of RNA aptamer and the pET-28a-Shig-amilCP plasmid, the expression of reporter gene amilCP was observed.**

To optimize the expression of reporter gene *amilCP* using the complementary fragment of aptamer and cell free system which is used for making hydrogel to detect *Shigella* in fridge, we prepared the cell free system extracted from BL21 cells transformed with pET-28a-*Shig-amilCP* plasmid. Add the complementary fragment of aptamer to the cell free system to observe the reporter gene expression.

The reaction conditions for expressing reporter protein in cell free system were optimized. The conditions including temperature, concentration of fragment, and reaction time were optimized in order to obtain sensitive and fast detection effect.

The result of optimization experiment was shown in Fig.14. The result shows that cell free system can express reporter protein in a wide range of temperatures, ranging from 4°C to 37°C, but the proper temperature varies from 25°C to 37°C for amilCP expression. The concentration of complementary fragment also has effect on reaction, and the lowest good concentration is about 2 uM. For visible reporter protein expression, it needed 6 h at least in cell free systems.

![](https://static.igem.wiki/teams/5074/eng-fig-14.jpg)

**Fig.14 The optimization result of reporter gene expression in cell free system transformed with pET-28a-*Shig-amilCP* after adding the complementary fragment of aptamer.**
(A) temperature,1: 4℃, 2: 10℃, 3: 15℃, 4: 20℃, 5: 25℃, 6:30℃, 7:37℃; (B) concentration,1: 0.5 uM, 2: 1 uM, 3: 2 uM, 4: 4 uM, 5: 6 uM, 6:8 uM, 7: 10 uM; (C) reaction time, 1:6 h, 2: 8 h, 3: 10 h, 4: 13 h, 5: 16 h, 6: 20 h, 7: 24 h.

**Summary:**

We successfully constructed the plasmid pET-28a-*Shig-amilCP*, which can express reporter gene *amilCP* under the control of complementary fragment of *Shigella* aptamer. At the presence of complementary fragment of *Shigella* aptamer, *amilCP* was expressed well. The optimized conditions to express reporter gene in cell free system are 25℃, 2 uM complementary fragment of aptamer, at least reaction 6 h.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/engineering#early)
**Return to index**

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}**List some sequences and protocols which are used in our experiments.**{% endblock %}
{% block page\_content %}

#### Index

* [###### Sequences information](https://2024.igem.wiki/icjfls/experiments#heading-2)

* [###### PCR protocol](https://2024.igem.wiki/icjfls/experiments#heading-3)

* [###### Gel electrophoresis](https://2024.igem.wiki/icjfls/experiments#heading-4)

* [###### Competent cell preparation](https://2024.igem.wiki/icjfls/experiments#heading-5)

* [###### Plasmid transforming](https://2024.igem.wiki/icjfls/experiments#heading-6)

* [###### Plasmid isolation](https://2024.igem.wiki/icjfls/experiments#heading-7)

* [###### Restriction endonuclease digestion](https://2024.igem.wiki/icjfls/experiments#heading-8)

* [###### Homologous recombination](https://2024.igem.wiki/icjfls/experiments#heading-9)

* [###### Protein purification](https://2024.igem.wiki/icjfls/experiments#heading-10)

* [###### SDS gel electrophoresis](https://2024.igem.wiki/icjfls/experiments#heading-11)

* [###### Cell lysate extract](https://2024.igem.wiki/icjfls/experiments#heading-12)

* [###### Cell-free system construction](https://2024.igem.wiki/icjfls/experiments#heading-13)

* [###### Hydrogel cell-free system](https://2024.igem.wiki/icjfls/experiments#heading-14)

## Overview

---

In our project, we have developed a biosensor using aptamer to detect *Shigella dysenteriae*, *Escherichia coli*, and *Salmonella typhimurium* in refrigerators. The experimental protocals are shown as follows.

## Obtain the relevant sequences information for this project

---

By querying databases, plasmid sequences, and literature references, we obtained the related sequences information as follows:

1. *AmilGFP* gene sequence：
ATGTCTTATTCAAAGCATGGCATCGTACAAGAAATGAAGACGAAATACCATATGGAAGGCAGTGTCAATGGCCATGAA TTTACGATCGAAGGTGTAGGAACTGGGTACCCTTACGAAGGGAAACAGATGTCCGAATTAGTGATCATCAAGCCTGCG GGAAAACCCCTTCCATTCTCCTTTGCATACTGTCATCAGTCTTTCAATATGGAAACCGTTGCTTCACAAAGTACCCGGCA GACATGCCTGACTATTTCAAGCAAGCATTCCCAGATGGAATGTCATATGAAAGGTCATTTCTATTTGAGGATGGAGCAG TTGCTACAGCCAGCTGGAACATTCGTCTCGAAGGAAATTGCTTCATCCACAAATCCATCTTTCATGGCGTAAACTTTCC CGCTGATGGACCCGTAATGAAAAAGAAGACAATTGACTGGGATAAGTCCTTCGAAAAAATGACTGTGTCTAAAGAGG TGCTAAGAGGTGACGTGACTATGTTTCTTATGCTCGAAGGAGGTGGTTCTCACAGATGCCAATTTCACTCCACTTACAA AACAGAGAAGCCGGTCACACTGCCCCCGAATCATGTCGTAGAACATCAAATTGTGAGGACCGACCTTGGCCAAAGTG TGCAAAAGGCTTTACAGTCAAGCTGGAAGCACATGCCGCGGCTCATGTTAACCCTTTGAAGGTTAAATAATAA

2. *mRFP* gene sequence：
ATGTCAGTGATTAAGCAGGTAATGAAGACCAAGTTGCACCTTGAGGGCACTGTCAATGGCCATGATTTTACGATCGA GGAAAATGACAGTCACCAAAGGCGCGCCTCTGCCGTTTTCCGTTCATATTCTTACACCTAGCCACATGTATGGAAGC AAACCGTTTAATAAGTATCCAGCGGATATCCCAGACTACCACAAACAGTCTTTTCCCGAAGGTATGTCTTGGGAGCG GTCGATGATTTTTGAAGATGGTGGCGTATGCACCGCCAGTAATCACTCCAGCATAAACTTGCAAGAGAACTGTTTCAT CTATGATGTTAAATTTCATGGTGTGAACCTGCCTCCGGATGGGCCCGTAATGCAAAAAACCATTGCTGGATGGGAGC CGAGCGTGGAAACACTGTACGTGCGTGACGGGATGTTAAAAAGTGACACTGCAATGGTTTTTAAACTGAAAGGAGG CGGTCATCATCGTGTTGATTTCAAAACGACGTATAAAGCCAAAAAACCTGTCAAGCTGCCAGAATTTCATTTCGTTGA ACATCGCCTGGAACTGACCAAACACGATAAAGATTTCACAACTTGGGACCAGCAGGAGGCAGCCGAAGGCCATTT CTCACCGCTGCCGAAGGCTCTCCCATAATAA

3. *amilCP* gene sequence：
ATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGACACTACTTTGAGGTCGAA GGCGATGGAAAAGGTAAGCCCTACGAGGGGGAGCAGACGGTAAAGCTCACTGTCACCAAGGGCGGACCTCTGCCA TTTGCTTGGGATATTTTATCACCACAGTGTCAGTACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGACT ATGTAAAGCAGTCATTCCCGGAGGGCTATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGTACTGTCA GCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACCATGTCAAGTTCTCTGGTTTGAACTTTCCTCCCAATGGACCT GTCATGCAGAGAAGACACAGGGCTGGGAACCCAACACTGAGCGTCTCTTTGCACGAGATGGAATGCTGCTAGGAAA CAACATTTATGGCTCTGAAGTTAGAAGGAGGCGGTCACTATTTGTGTGAATTTAAAACTACTTACAAGGCAAAGAAGT GAACCTGTGAAGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAATCACAACAAGGATTACACTTCG GTTGAGCAGTGATTTCCATTGCACGCAAACCTGTGGTCGCCTAATAA

4. The aptamer sequence of *Shigella dysenteriae*:
CGGAACUAGCGUUUAAAUGCCAGGACUGAAGUAGGCAGGG

5. The aptamer sequence of *Escherichia coli*:
AUACCAGCUUAUUCAAUUGCACGAAUUUGCUGUGUUUUUGGGGGGGUCGGGGAGUAUAAGAUAGUAAGUG
CAAUCU

6. The aptamer sequence of *Salmonella typhimurium*:
UCACUGUUAUCCGAUAGCAGCGCGGGAUGA

7.The *lacZ* gene sequence is from NC\_000913.3:c366305-363231 *Escherichia coli* str. K-12 substr. MG1655.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/experiments#early)
**Return to index**

## PCR protocol

---

The reaction components are assembled in the PCR protocol as described below. The final volume should be 20 µL.
1. All reagents are thawed on ice.
2. Assemble reaction mix into 20 µL volume in a thin walled 0.2 mL PCR tubes as follow:
    8 uL ddH2O
    1 µL DNA Template
    0.5 uL Primer F
    0.5 uL Primer R
    10 uL 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.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/experiments#early)
**Return to index**

## Gel Electrophoresis

---

1. Prepare 100mL 1xTAE 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 1x TAE buffer.
5. Load 8 μL of DNA maker into lane.
6. Mix 1 μL of 10x 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.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/experiments#early)
**Return to index**

## 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 3 mL of LB broth solution. Incubate it at 37°C overnight (more than 16 h) with shaking at 220 rpm.
3. Transfer 250 uL 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 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 8000 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 uL per tube. Now the competent cells are ready to be transformed.
Note: Transformation and Storage Solution (TSS) contains LB medium 85% 8.5 mL, PEG (wt/vol, Mw 8000) 10% 1.0 g, DMSO (vol/vol) 5% 500 μL, and MgCl2 (pH 6.5) 50 mM 500 μL.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/experiments#early)
**Return to index**

## Plasmid Transforming and Pick monoclonal

---

1. Thaw TSS cells on ice.
2. Add 5 uL 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 uL 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.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/experiments#early)
**Return to index**

## Plasmid Isolation Protocol

---

1. The bacteria that contained target plasmid were cultivated at 37°C overnight.
2. Take out 1,500 uL solution of cultivated bacteria to a 2mL 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 uL 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 uL 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 uL 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 uL 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 uL 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 uL 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.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/experiments#early)
**Return to index**

## 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 (*Eco*R1 and *Hin*dⅢ)
    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.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/experiments#early)
**Return to index**

## 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 BL21D*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.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/experiments#early)
**Return to index**

## 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-*lacZ* 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.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/experiments#early)
**Return to index**

## SDS Gel 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.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/experiments#early)
**Return to index**

## Preparation of *E. coli* BL21D*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 10000 g 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 8000 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 2S, turn off for 2S, the total time is 15 min, and the temperature alarm is set at 40°C.
7. Centrifuge the tube at 4°C, 12000 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.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/experiments#early)
**Return to index**

## 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* BL21D*LacZ* cell lysate extract, 5% (V/V) 20 mg/mL X-gal chromogenic substrate.
3. Add 5 nM pET-28a-aptamer-*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.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/experiments#early)
**Return to index**

## Preparation of hydrogel cell-free system

---

1. Prepare 0.25% (w/v) initiator standard solution. Take 20 mL PBS and add it to a brown bottle containing 0.05g LithiumPhenyl (2,4,6-trimethylbenzoyl) phosphinate (LAP); Heat and dissolve in a 45°C water bath for 15 min, oscillating several times during this period.
2. Prepare Gelatin Methacryloyl (GelMA) solution (GelMA concentration of 5% (w/v)). Take the required mass of GelMA and place it in a centrifuge tube; Take the initiator standard solution mentioned above and add it to the centrifuge tube, shake it to fully lubricate GeIMA; Dissolve in a 65°C water bath and avoid light for 30 min, oscillating several times during this period until completely dissolved; Centrifuge to remove bubbles from the solution (3000 rpm. 2 min); Immediately use 0.22 um sterile needle filter to sterilize GelMA solution.
3. Preparation of cell-free hydrogel. Take a cell-free system and suspend it in GelMA solution preheated at 37°C to prepare a cell-free system suspension; Inject it into the 24 hole plate (300-500 uL/hole) of the orifice plate, irradiate the gel with 405 nm light source for 10-30 sec, and adjust the gel strength through the light time and intensity.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/experiments#early)
**Return to index**

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}**Show some achievements collected from the wet laboratory for our project!**{% endblock %}
{% block page\_content %}

#### Index

* [###### Construct positive control plasmids](https://2024.igem.wiki/icjfls/results#heading-1)

* [###### Constuct aptamer toehold switch plasmids](https://2024.igem.wiki/icjfls/results#heading-2)

* [###### Transformed with aptamer toehold switch plasmids](https://2024.igem.wiki/icjfls/results#heading-3)

* [###### Optimize cell free expression systems](https://2024.igem.wiki/icjfls/results#heading-4)

* [###### Construct aptamer toehold *LacZ* plasmids](https://2024.igem.wiki/icjfls/results#heading-5)

* [###### Construct BL21 cells lacking of *LacZ* gene](https://2024.igem.wiki/icjfls/results#heading-6)

* [###### Optimize cell free System expressing *LacZ* Gene](https://2024.igem.wiki/icjfls/results#heading-7)

* [###### Experiment on cell free system hydrogel](https://2024.igem.wiki/icjfls/results#heading-8)

* [###### Conclusion](https://2024.igem.wiki/icjfls/results#heading-9)

* [###### Future plan and consideration](https://2024.igem.wiki/icjfls/results#heading-10)

![](https://static.igem.wiki/teams/5074/heyingshiyanshi.jpg)

## Overview

---

In our project, we developed a biosensor to detect *Shigella*, *Salmonella* and *Escherichia coli* in refrigerator, based on nucleic acid aptamers, toehold switch and cell free hydrogel. These bacteria present in refrigerator are big threat to human health. If we can detect bacteria in the refrigerator in a timely manner and send out a warning, reminding people to clean and disinfect the refrigerator in time, it will be beneficial to protect people's physical health. Here, we show the main results of the experiments which are the basement of the biosensor constructed in our project.

## 1. Construction and identification of positive control plasmids

---

In order to make the detection of pathogenic bacteria more intuitive and visible, we used some chromoproteins as the reporters. We constructed 3 positive control plasmids containing 3 different chromoprotein genes.

One of them is *amilCP* gene (part K592009), of which protein shows purple color. The gene of *amilCP* was amplified using PCR method and inserted into the vector pET-28a (5362 bp). The construction of the plasmid pET-28a-*amilCP* was identified by restriction endonuclease digestion with *Eco*R I and *Hin*d III according to the sequence. The length of *amilCP* gene is about 701 bp. Through agarose electrophoresis, we found that the lengths of digested fragments are consistent with the expected result (Fig. 1A).

In order to test whether the constructed plasmid pET-28a-*amilCP* express the chromoprotein, it was transformed into the BL21 strain. Then the transformed cells were plated on the agar. Some purple clones were shown in the plate (Fig. 1B), illustrating that the positive control plasmid pET-28a-*amilCP* was constructed well and expressed amilCP normally. It was used as positive control for the construction of subsequent RNA toehold switch plasmid.

![](https://static.igem.wiki/teams/5074/r-fig-1-the-identification-of-pet-28a-amilcp-plasmid.jpg)

**Fig. 1 The identification of pET-28a-*amilCP* plasmid**
(A) Electrophoresis result of plasmid digested by restriction endonucleases. M: Marker, 1: pET-28a-*amilCP* plasmid, 2: The plasmid digested by *Eco*R I and *Hin*d III restriction endonucleases. (B) Expression of *amilCP* gene in BL21 cells transformed with pET-28a-*amilCP* plasmid.

Another reporter gene is *mRFP* (part J04450), of which protein shows red color. The gene of *mRFP* was amplified using PCR method and inserted into the expression vector pET-28a (5362 bp). The construction of the plasmid pET-28a-mRFP was also identified by restriction endonuclease digestion with *Eco*R I and *Hin*d III. The length of mRFP gene is 714 bp. Through agarose electrophoresis, we found that the lengths of digested fragments are consistent with the expected result (Fig. 2A).

In order to test whether the constructed plasmid pET-28a-*mRFP* express the chromoprotein, it was transformed into the BL21 strain. Then the transformed cells were plated on the agar. Some red clones were shown in the plate (Fig. 2B), illustrating that the positive control plasmid pET-28a-*mRFP* was constructed well and expressed normally. It was used as positive control for the construction of subsequent RNA toehold switch plasmid.

![](https://static.igem.wiki/teams/5074/r-fig-2-the-identification-of-pet-28a-mrfp-plasmid.jpg)

**Fig. 2 The identification of pET-28a-*mRFP* plasmid**
(A) Electrophoresis result of plasmid digested by restriction endonucleases. M: Marker, 1: pET-28a-*mRFP* plasmid, 2: The plasmid digested by *Eco*R I and *Hin*d III restriction endonucleases. (B) Expression of *mRFP* gene in BL21 cells transformed with pET-28a-*mRFP* plasmid.

The third reporter gene is *EGFP* (part E0040), of which protein shows florescent green color. The gene of *EGFP* was amplified using PCR method and inserted into the expression vector pET-28a (5362 bp). The construction of the plasmid pET-28a-*EGFP* was also identified by restriction endonuclease digestion with *Eco*R I and *Hin*d III. The length of EGFP gene is 704 bp. Through agarose electrophoresis, we found that the lengths of digested fragments are consistent with the expected result (Fig. 3A).

In order to test whether the constructed plasmid pET-28a-*EGFP* express the florescent protein, it was transformed into the BL21 strain. Then the transformed cells were plated on the agar. Some green clones were shown in the plate (Fig. 3B), illustrating that the positive control plasmid pET-28a*-EGFP* was constructed well and expressed normally. It was used as positive control for the construction of subsequent RNA toehold switch plasmid.

![](https://static.igem.wiki/teams/5074/r-fig-3-the-identification-of-pet-28a-egfp-plasmid.jpg)

**Fig. 3 The identification of pET-28a-*EGFP* plasmid**
(A) Electrophoresis result of plasmid digested by restriction endonucleases. M: Marker, 1: pET-28a-*EGFP* plasmid, 2: The plasmid digested by *Eco*R I and *Hin*d III restriction endonucleases. (B) Expression of *EGFP* gene in BL21 cells transformed with pET-28a-*EGFP* plasmid.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/results#early)
**Return to index**

## 2. Construction and identification of aptamer toehold switch plasmids

---

According to the aptamer sequence of *Shigella* (please refer to [Experiments](https://2024.igem.wiki/icjfls/experiments) section for details) and the construction rules of toehold switch, we designed the aptamer toehold switch with *amilCP* reporter gene, which was aimed to detect *Shigella*. The fragment of aptamer toehold switch was synthesized and inserted into pET-28a-*amilCP* plasmid using PCR method. This plasmid was named as pET-28a-*Shig*-*amilCP* which was identified by restriction endonucleases (*Eco*R I and *Hin*d III) digestion assays. The inserted fragment length was about 801 bp. The agarose electrophoresis result showed that the inserted fragment length was consistent with the expected result (Fig. 4A).

Then the constructed plasmid was transformed into BL21 cells, and the result showed that no purple clone was observed (Fig. 4B), illustrating the aptamer toehold switch plasmid was constructed successfully, which purple protein didn’t express because of the translation inhibition by aptamer toehold switch.

![](https://static.igem.wiki/teams/5074/r-fig-4-identification-of-pet-28a-shig-amilcp-aptamer-toehold-plasmid.jpg)

**Fig. 4 Identification of pET-28a-*Shig-amilCP* aptamer toehold switch plasmid.**
(A) Electrophoresis result of plasmid and PCR. M: Marker, 1: Plasmid of pET-28a-*Shig-amilCP*, 2: The PCR result of aptamer toehold switch *amilCP*. (B) No purple clone was observed in BL21 cells transformed with pET-28a-*Shig-amilCP* plasmid.

According to the aptamer sequence of *E. coli* (please refer to [Experiments](https://2024.igem.wiki/icjfls/experiments) section for details) and the construction rules of toehold switch, we designed the aptamer toehold switch with *mRFP* reporter gene, which was aimed to detect *E. coli*. The fragment of aptamer toehold switch was synthesized and inserted into pET-28a-*mRFP* plasmid using PCR method. This plasmid was named as pET-28a-*Esch-mRFP* which was identified by restriction endonucleases (*Eco*R I and *Hin*d III) digestion assays. The inserted fragment length was about 850 bp. The agarose electrophoresis result showed that the inserted fragment length was consistent with the expected result (Fig. 5A).

Then the constructed plasmid was transformed into BL21 cells, and the result showed that no red clone was observed (Fig. 5B), illustrating the aptamer toehold switch plasmid was constructed successfully, which red protein didn’t express because of the translation inhibition by aptamer toehold switch.

![](https://static.igem.wiki/teams/5074/r-fig-5-identification-of-pet-28a-esch-mrfp-aptamer-toehold-plasmid.jpg)

**Fig. 5 Identification of pET-28a-*Esch-mRFP* aptamer toehold switch plasmid.**
(A) Electrophoresis result of plasmid and PCR. M: Marker, 1: Plasmid of pET-28a-*Esch-mRFP*, 2: The PCR result of aptamer toehold switch *mRFP*. (B) No purple clone was observed in BL21 cells transformed with pET-28a-*Esch-mRFP* plasmid.

According to the aptamer sequence of *Salmonella* (please refer to [Experiments](https://2024.igem.wiki/icjfls/experiments) section for details) and the construction rules of toehold switch, we designed the aptamer toehold switch with *EGFP* reporter gene, which was aimed to detect *Salmonella*. The fragment of aptamer toehold switch was synthesized and inserted into pET-28a-*EGFP* plasmid using PCR method. This plasmid was named as pET-28a-*Salm-EGFP* which was identified by restriction endonucleases (*Eco*R I and *Hin*d III) digestion. The inserted fragment length was about 794 bp. The agarose electrophoresis result showed that the inserted fragment length was consistent with the expected result (Fig. 6A).

Then the constructed plasmid was transformed into BL21 cells, and the result showed that no green clone was observed (Fig. 6B), illustrating the aptamer toehold switch plasmid was constructed successfully, which florescent protein didn’t express because of the translation inhibition by aptamer toehold switch.

![](https://static.igem.wiki/teams/5074/r-fig-6-identification-of-pet-28a-salm-egfp-aptamer-toehold-plasmid.jpg)

**Fig. 6 Identification of pET-28a-*Salm-EGFP* aptamer toehold switch plasmid.**
(A) Electrophoresis result of plasmid and PCR. M: Marker, 1: Plasmid of pET-28a-*Salm-EGFP*, 2: The PCR result of aptamer toehold switch *EGFP*. (B) No green clone was observed in BL21 cells transformed with pET-28a-*Salm-EGFP* plasmid.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/results#early)
**Return to index**

## 3. Experiments of BL21 cells transformed with aptamer toehold switch plasmids

---

To study whether the aptamer toehold switches work or not, 3 complementary fragments of RNA aptamers of *Shigella*, *E. coli* and *Salmonella* were synthesized. Then both the aptamer toehold switch plasmids and their corresponding complementary fragments of RNA aptamers were co-transformed into the BL21 cells. The expression of reporter genes was observed (Fig.7), indicating that the aptamer toehold switches can control the expression of reporter proteins, and the complementary fragments of RNA aptamers can trigger the expression of corresponding reporter gene.

![](https://static.igem.wiki/teams/5074/r-fig-7-the-expression-of-reporter-proteins-in-transformed-cells.jpg)

**Fig.7 The expression of reporter genes in co-transformed cells.**
(A) Reporter protein (amilCP) expressed in cells co-transformed with pET-28a-*Shig-amilCP* plasmid and corresponding complementary fragment of *Shigella* aptamer; (B)Reporter protein (EGFP) expressed in cells co-transformed with pET-28a-*Salm-EGFP* plasmid and corresponding complementary fragment of *Salmonella* aptamer; (C)Reporter protein (mRFP) expressed in cells co-transformed with pET-28a-*Esch-mRFP* plasmid and corresponding complementary fragment of *E. coli* aptamer.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/results#early)
**Return to index**

## 4. Optimization of reporter gene expression in cell free systems

---

To optimize the expression of reporter genes using the complementary fragments of aptamers and cell free systems which are used for making hydrogel to detect bacteria in fridge, we prepared the cell free systems extracted from BL21 cells transformed with constructed plasmids. After culturing the transformed cells for about 16 h, the cells were extracted to obtain cell extracts (please refer to [Experiments](https://2024.igem.wiki/icjfls/experiments)  section for details). Add some nutrient components to the cell extracts for getting cell free systems (please refer to [Experiments](https://2024.igem.wiki/icjfls/experiments)  section for details), then add the complementary fragments of aptamer of *Shigella*, *E. coli* and *Salmonella* to the corresponding 3 cell free systems, respectively.

The reaction conditions for expressing reporter proteins in cell free system were optimized using the synthesized complementary fragments of aptamers. The conditions including temperature, concentration of fragment, and reaction time were optimized in order to obtain sensitive and fast detection effects. The results of optimization experiment were showed in Fig. 8-Fig. 10.

Temperature is a primary factor affecting protein expression in vitro, and the optimal temperature for different proteins to express may be different. The optimization results showed that cell free system can express reporter proteins in a wide range of reaction temperatures, ranging from 4°C to 37°C, but the proper temperature varies from 25°C to 37°C for reporter proteins. The concentration of complementary fragment also has effect on reaction, and the lowest good concentration is about 2 uM. For visible reporter protein expression, it needed 6 h at least in cell free systems.

![](https://static.igem.wiki/teams/5074/r-fig-8-the-optimization-results-of-reporter-gene-expression-in-cell-free-system-transformed-with-pet-28a-shig-amilcp-after-adding-its-corresponding-complementary-fragment-of-aptamer.jpg)

**Fig. 8 The optimization results of reporter gene expression in cell free system transformed with pET-28a-*Shig-amilCP* after adding its corresponding complementary fragment of aptamer.**
(A) temperature,1: 4℃, 2: 10℃, 3: 15℃, 4: 20℃, 5: 25℃, 6: 30℃, 7: 37℃; (B) concentration,1: 0.5uM, 2: 1uM, 3: 2uM, 4: 4uM, 5: 6uM, 6: 8uM, 7: 10uM; (C) reaction time, 1: 6h, 2: 8h, 3: 10h, 4: 13h, 5: 16h, 6: 20h, 7: 24h.

![](https://static.igem.wiki/teams/5074/r-fig-9-the-optimization-results-of-reporter-gene-expression-in-cell-free-system-transformed-with-pet-28a-esch-mrfp-after-addingits-corresponding-complementary-fragment-of-aptamer.jpg)

**Fig.9 The optimization results of reporter gene expression in cell free system transformed with pET-28a-*Esch-mRFP* after adding its corresponding complementary fragment of aptamer.**
(A) temperature,1: 4℃, 2: 10℃, 3: 15℃, 4: 20℃, 5: 25℃, 6: 30℃, 7: 37℃; (B) concentration,1: 0.5uM, 2: 1uM, 3: 2uM, 4: 4uM, 5: 6uM, 6: 8uM, 7: 10uM; (C) reaction time, 1: 6h, 2: 8h, 3: 10h, 4: 13h, 5: 16h, 6: 20h, 7: 24h.

![](https://static.igem.wiki/teams/5074/r-fig-10-the-optimization-results-of-reporter-gene-expression-in-cell-free-system-transformed-with-pet-28a-salm-egfp-after-adding-its-corresponding-complementary-fragment-of-aptamer.jpg)

**Fig.10 The optimization results of reporter gene expression in cell free system transformed with pET-28a-*Salm-EGFP* after adding its corresponding complementary fragment of aptamer..**
(A) temperature,1: 4℃, 2: 10℃, 3: 15℃, 4: 20℃, 5: 25℃, 6: 30℃, 7: 37℃; (B) concentration,1: 0.5uM, 2: 1uM, 3: 2uM, 4: 4uM, 5: 6uM, 6: 8uM, 7: 10uM; (C) reaction time, 1: 6h, 2: 8h, 3: 10h, 4: 13h, 5: 16h, 6: 20h, 7: 24h.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/results#early)
**Return to index**

## 5. Construction and identification of pET-28a-aptamer toehold *LacZ* plasmid

---

In order to improve sensitivity and reduce detection time, the report genes we used above were replaced with *LacZ* gene which can express an enzyme, amplifying the detection result. So, the pET-28a-*Shig-lacZ*, pET-28a-*Esch-lacZ*, and pET-28a-*Salm-lacZ* toehold switch plasmids were constructed using *lacZ* as reporter gene, which were used to detect *Shigella*, *E. coli* and *Salmonella*, respectively, in cell free systems. *lacZ* can express β-galactosidase, catalyzing the blue product generation using X-gal, which amplifies the detection results.

For identification of these plasmids, the restriction endonuclease digestion and PCR assay were performed. The results showed that the fragment lengths of aptamer toehold switch with *lacZ* were consistent with the expected results, indicating that pET-28a-*Shig-LacZ*, pET-28a-*Esch-LacZ* and pET-28a-*Salm-LacZ* plasmids were constructed successfully (Fig. 11 A, B and C).

![](https://static.igem.wiki/teams/5074/r-fig-11-identification-of-aptamer-toehold-plasmids-with-lacz-gene.jpg)

**Fig.11 Identification of aptamer toehold switch with *LacZ* gene plasmids.**
(A) pET-28a-*Shig-LacZ* plasmid. M: Marker, 1: The plasmid of pET-28a-*Shig-LacZ*, 2: PCR result, 3: Digestion result by *Eco*R I and *Hin*d III restriction endonuclease. (B) pET-28a*-Esch-LacZ* plasmid. M: Marker, 1: The plasmid of pET-28a-*Esch-LacZ*, 2: PCR result, 3: Digestion result by *Eco*R I and Hind III restriction endonuclease. (C) pET-28a-*Salm-LacZ* plasmid. M: Marker, 1: The plasmid of pET-28a-*Salm-LacZ*, 2: PCR result, 3: Digestion result by *Eco*R I and *Hin*d III restriction endonuclease.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/results#early)
**Return to index**

## 6. Construction of BL21 cells lacking of *LacZ* gene

---

In order to eliminate the influence of the BL21 cells’ own β-galactosidase, we constructed BL21 mutant strain with *LacZ* gene deletion (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 (1.6 kb) was obtained by PCR amplification (Fig. 12). This PCR product of *kanamycin* gene was flanked with the start and end sequences of *LacZ* gene.

![](https://static.igem.wiki/teams/5074/r-fig-12-the-pcr-result-of-kanamycin-gene-using-pet-28a-vector-as-a-template.jpg)

**Fig. 12 The PCR result of *Kanamycin* gene using pET-28a vector as a template.**
M: Marker, 1: *Kanamycin* gene amplified by PCR method.

The PCR product of amplified *kanamycin* gene flanked with the start and end sequences of *LacZ* gene was transformed into BL21 strain in which homologous recombination occurred between the PCR product and the *LacZ* gene of BL21 genome, and BL21 mutant strain with LacZ gene deletion (BL21D*LacZ*) was obtained. This BL21D*LacZ* strain was screened out with kanamycin and confirmed by PCR results of *LacZ* and *kanamycin* genes (Fig. 13), indicating that the *LacZ* gene deletion BL21 strain (BL21D*LacZ*) with kanamycin resistant was constructed successfully.

![](https://static.igem.wiki/teams/5074/r-fig-13-the-pcr-results-of-kanamycin-and-lacz-genes-in-kanamycin-resistant-bl21dlacz-strain.jpg)

**Fig. 13 The PCR results of *Kanamycin* and *LacZ* genes in kanamycin resistant BL21D*LacZ* strain.**
M: Marker, 1: The amplification of *Kanamycin* gene, 2: The amplification of *LacZ* gene (No band).

To identify whether the *LacZ* gene of recombinant plasmid express or not，both the complementary fragment of RNA aptamer and the plasmid pET-28a-*Esch-lacZ* were transformed into BL21D*LacZ* strain. After culture 12 h with fresh LB medium containing Kanamycin and Ampicillin, the LacZ protein (β-galactosidase) was purified using 6x His tag column for identification. The SDS-PAGE electrophoresis result was shown in Fig.14.

![](https://static.igem.wiki/teams/5074/r-fig-14-the-sds-page-result-shows-expression-and-purification-of-lacz-proteins.jpg)

**Fig. 14 The SDS-PAGE result shows expression and purification of LacZ protein.**
M: Marker, 1: All supernatant proteins of BL21D*LacZ* strain, 2: All supernatant proteins of BL21D*LacZ* strain transformed with pET-28a-*Esch-LacZ* and the complementary fragment of *E. coli* aptamer, 3: Purified LacZ protein from the supernatant proteins of sample 2.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/results#early)
**Return to index**

## 7. Experiment performed in cell free system using *LacZ* gene as reporter

---

Due to concerns about the pathogenicity and safety, we did not conduct the detection experiments using bacteria. We only used the complementary fragment of *E. coli* aptamer to show the feasibility of our constructed cell free system containing pET-28a*-Esch-LacZ* plasmid for bacteria detection. The cell free system was prepared using plasmid pET-28a*-Esch-LacZ* transformed BL21D*LacZ* strain. Complementary fragment and X-gal (substrate of β-galactosidase) were added to the cell free system under different conditions including temperature, concentration of complementary fragment, concentration of X-gal, and detection time. Since the maximum absorbance of the blue product is located at 615 nm, the experiment results were quantified using absorbance value, which were showed in Fig. 15. The results indicated that the cell free system works well. For intuitive and visible good result, the best conditions for reaction are 20℃, 0.5 uM complementary fragment of aptamer, 100 ug/mL X-gal, and 1 h reaction time.

![](https://static.igem.wiki/teams/5074/r-fig-15the-quantification-result-of-experiment-performed-in-cell-free-system-containing-pet-28a-esch-lacz-plasmidunder-different-conditions.jpg)

**Fig.15 The quantification result of experiment performed in cell free system containing pET-28a-*Esch-LacZ* plasmid under different conditions.**
(A) Absorbance of the end product catalyzed by β-galactosidase expressed in cell free system with 1 uM complementary fragment of aptamer and 100 ug/mL X-gal, 1 h reaction time, under different temperature; (B) Absorbance of the end product catalyzed by β-galactosidase expressed in cell free system with 100 ug/mL X-gal and different concentration of complementary fragment of aptamer,1 h reaction time, under 20℃; (C) Absorbance of the end product catalyzed by β-galactosidase expressed in cell free system with1 uMcomplementary fragment of aptamer and different concentration of X-gal, 1 h reaction time, under 20℃; (D) Absorbance of the end product catalyzed by β-galactosidase expressed in cell free system with 1 uM complementary fragment of aptamer and 100 ug/mL X-gal, different reaction time, under 20℃.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/results#early)
**Return to index**

## 8. Experiment on cell free system hydrogel

---

Hydrogel is a three-dimensional (3D) network polymer with hydrophilic groups, which can absorb moisture but are insoluble in water. Hydrogel is widely used in various fields due to its unique physical and chemical properties. It is suitable for the three-dimensional structure of cell growth and differentiation. In addition, hydrogel has good mechanical property.

In our experiment, we combined the cell free system with methacrylated gelatin (GelMA) hydrogel which has excellent biocompatibility and cell reaction characteristics, making high expression ability of cell free system and controllable catalyzing reaction. This combination made the possibility of bacteria detection in fridge using cell free system. So, the cell free system hydrogel was constructed using cell free system extracted from BL21D*LacZ* strain transformed with pET-28a-*Esch-LacZ* plasmid and hydrogel ( GelMA).

Add 0.5 uM or 1 uM complementary fragment of aptamer and 100 ug/mL X-gal onto the GelMA hydrogel containing cell free system, reaction 2 h under 4℃. The result was showed in Fig. 16, indicating that it is possible using the cell free system hydrogel to detect bacteria in refrigerator.

![](https://static.igem.wiki/teams/5074/r-fig-16-experiment-on-cell-free-system-hydrogel-reaction-2h-under-4.jpg)

**Fig. 16 Experiment on cell free system hydrogel, reaction 2 h, under 4℃.**
(A) The negative control without complementary fragment of aptamer or X-gal; (B) The result using 0.5 uM complementary fragment of aptamer and 100 ug/mL X-gal; (C) The result using 1 uM complementary fragment of aptamer and 100 ug/mL X-gal.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/results#early)
**Return to index**

## 9. Conclusion

---

We constructed a biosensor using cell free system hydrogel containing RNA toehold switch, which is the main component of a detector used for detecting pathogenic bacteria in refrigerator. When the bacteria fall to the hydrogel in fridge, it will switch on the RNA toehold to express *lacZ* gene and produce β-galactosidase, catalyzing the blue product which is detected by the biosensor. This information is scanned and transferred by RGB color sensor to the pilot lamp, making the red LED light up on the screen of refrigerator door, reminding people to clean or disinfect the refrigerator in time. It is very convenient and beneficial for the people to use it in refrigerator at home.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/results#early)
**Return to index**

## 10. Future plan and consideration

---

Due to the low temperature of the refrigerator, the enzymatic reaction occurring on the biosensor is relatively slow. Therefore, in future research and development, we will continuously optimize the conditions of enzymatic reaction to increase enzyme activity, achieving better detection results. Or we may search for an enzyme that can tolerate low temperature, to instead the current enzyme (β-galactosidase).

In addition, it is necessary to continuously increase the number of bacteria aptamers for detecting more pathogens and broadening the bacteria range of detection.

Most data in our project were rough due to the time limitation and our ability. Some experiments need to be replicated and modified for obtaining reliable results in the future.

[![](https://static.igem.wiki/teams/5074/return-to-index.jpg)](https://2024.igem.wiki/icjfls/results#early)
**Return to index**

{% endblock %}