Difference between revisions of "Part:BBa K5074001"

Latest revision as of 11:02, 2 October 2024

AmilCP generator controlled by Shigella RNA aptamer toehold switch

It comprises Shigella dysenteriae RNA aptamer, RBS, Linker and reporter gene amilCP. 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 deteced. It is used to detect the bacteria Shigella in refrigerator.

Sequence and Features

In order to make the detection of pathogenic bacteria more intuitive and visible, we used some chromoproteins as the reporters. One of which is AmilCP(K592009) which shows purple chromoprotein used to construct BBa_K5074001.

According to the aptamer sequence of Shigella dysenteriae and the construction rules of toehold switch, we designed the aptamer toehold switch with amilCP reporter gene, which was aimed to detect Shigella dysenteriae in refrigerator.

This part comprises Shigella dysenteriae RNA aptamer, RBS(B0034), Linker and reporter gene amilCP(K592009). It’s sequence was analyzed using Snapgene Viewer for restriction enzyme cleaving sites to congstruct the plasmid pET-28a-Shig-amilCP using the proper enzymes. The analysis result with Snapgene Viewer was shown in Fig.1.

Fig.1 The endonuclease map of BBa_K5074001 analized using SnapGene Viewer, showing the restriction enzyme information (no EcoRI and Hind III cleaving sites).

The sequence of aptamer toehold switch was synthesized and inserted into pET-28a-amilCP plasmid using PCR method. This plasmid was named as pET-28a-Shig-amilCP which was identified by PCR amplification. The inserted fragment length was about 801bp. The agarose electrophoresis result showed that the PCR product was consistent with the expected result (Fig.2).

Fig.2 Electrophoresis Identification of pET-28a-Shig-amilCP aptamer toehold switch plasmid with PCR method.

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.3A), illustrating the aptamer toehold switch plasmid was constructed successfully, which purple protein didn’t express because of the translation inhibition by aptamer toehold switch.

Fig.3 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.4), indicating that the aptamer toehold switch can control the expression of reporter gene, and the complementary fragment of RNA aptamer can trigger the expression of reporter gene.

Fig.4 After transformation of both complementary fragment of RNA aptamer and the pET-28a-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.5.

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 2uM. For visible reporter protein expression, it needed 6h at least in cell free systems.

Fig.5 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: 0.5uM, 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.

Summary:

We successfullly 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℃, 2uM complementary fragment of aptamer, at least reaction 6h.