Difference between revisions of "Part:BBa K3896016"

Latest revision as of 16:27, 20 October 2021

The gene of recombinant PmrCAB (DPP4) two-component and quorum sensing system

We replaced sensitive domains Trp34 to Glu64 of the original PmrB with the core binding domain of DPP4 protein Gly260 to Asp330 to be stimulated by the S protein of MERS-CoV. After detecting the S protein of MERS-CoV, the sensor kinase PmrB autophosphorylates the highly conserved histidine residue, and then transfers the phosphate group to the conserved aspartate residue in its homologous reaction regulator PmrA. Then phosphorylated PmrA protein combined with promoter PmrC sequence to activate the expression of reporter gene.

In order to improve the sensitivity of detection, we have improved the above parts and added a quorum sensing system (Fig. 1).

In the AHL mediated quorum sensing system composed of LuxI / LuxR, LuxI homologous gene in bacteria is responsible for the synthesis of QS signal molecule AHL, and LuxR homologue is activated as the receptor of this signal molecule to regulate the transcription of downstream genes. In this design, when the engineering bacteria recognize the virus S protein, PmrC starts the expression of the downstream gene LuxI, AHL began to synthesize continuously. When the concentration of AHL reached the threshold, it combined with LuxR protein to form LuxR-AHL complex and activated the corresponding promoter Plux_ HS,then activated the expression of reporter gene EGFP.

Fig.1 Schematic diagram of the overall design of quorum sensing system

In order to add the quorum sensing system to the detection system, we set up the following gene circuit and double transferred the two plasmids into E. coli BL21 (DE3)(Fig.2).

Fig.2 The two plasmids were constructed with pETDuet-1 and pACYCDuet-1 as the vectors, and were co-transformed to E.coli BL21(DE3) to verify the quorum sensing system

Overnight shaking bacteria carrying two plasmids from the medium plate containing two kinds of resistance, diluted the bacterial solution to OD = 2, and the final volume was 5ml. Then, when the bacterial solution grows to OD = 0.4 ~ 0.6, IPTG is added to induce the expression of PmrB and PmrA, and S protein is added. After PmrB feels the stimulation of S protein, PmrC is activated through a series of reactions to start the expression of LuxI and LuxR downstream. LuxI begins to continuously synthesize AHL signal molecules and enter the outside of the cell. After reaching a certain threshold, it can enter other engineering bacteria, Combined with endogenous LuxR to form a complex, activate the corresponding promoter Plux_HS and further express the downstream reporter gene EGFP.

We set up different experimental groups, and the results are as follows(Fig.3).

Fig.3 The fluorescence intensity of engineered bacteria without quorum sensing system and with quorum sensing

By joining the quorum sensing system. When a few engineering bacteria detect the virus, the information can be transmitted to other engineering bacteria through the mediation of signal molecule AHL, and finally the population expresses the reporter gene EGFP, which makes the detection results significant.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1900
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 3003
Illegal BamHI site found at 2328
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1147
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K3896016 short</partinfo>
-We replaced the Fe (III) sensitive domains Trp34 to Glu64 of the original PmrB with the core binding domain of DPP4 protein Gly260 to Asp330 to be stimulated by the S protein of MERS-CoV. After detecting the S protein of MERS-CoV, the sensor kinase PmrB autophosphorylates the highly conserved histidine residue, and then transfers the phosphate group to the conserved aspartate residue in its homologous reaction regulator PmrA. Then phosphorylated PmrA protein combined with promoter PmrC sequence to activate the expression of reporter gene.
+We replaced sensitive domains Trp34 to Glu64 of the original PmrB with the core binding domain of DPP4 protein Gly260 to Asp330 to be stimulated by the S protein of MERS-CoV. After detecting the S protein of MERS-CoV, the sensor kinase PmrB autophosphorylates the highly conserved histidine residue, and then transfers the phosphate group to the conserved aspartate residue in its homologous reaction regulator PmrA. Then phosphorylated PmrA protein combined with promoter PmrC sequence to activate the expression of reporter gene.
 In order to improve the sensitivity of detection, we have improved the above parts and added a quorum sensing system (Fig. 1).
-In the AHL mediated quorum sensing system composed of LuxI / LuxR, LuxI homologous gene in bacteria is responsible for the synthesis of QS signal molecule AHL, and LuxR homologue is activated as the receptor of this signal molecule to regulate the transcription of downstream genes. In this design, when the engineering bacteria recognize the virus S protein, PmrC starts the expression of the downstream gene LuxI, AHL began to synthesize continuously. When the concentration of AHL reached the threshold, it combined with LuxR protein to form LuxR-AHL complex and activated the corresponding promoter plux_ HS,then activated the expression of reporter gene EGFP.
+In the AHL mediated quorum sensing system composed of LuxI / LuxR, LuxI homologous gene in bacteria is responsible for the synthesis of QS signal molecule AHL, and LuxR homologue is activated as the receptor of this signal molecule to regulate the transcription of downstream genes. In this design, when the engineering bacteria recognize the virus S protein, PmrC starts the expression of the downstream gene LuxI, AHL began to synthesize continuously. When the concentration of AHL reached the threshold, it combined with LuxR protein to form LuxR-AHL complex and activated the corresponding promoter Plux_ HS,then activated the expression of reporter gene EGFP.
-figure 1 (QSpicture)
+https://static.igem.org/mediawiki/parts/0/03/T--NEU_CHINA--daaaaaa.png
-In order to add the quorum sensing system to the detection system, we set up the following gene circuit and double transferred the two plasmids into E. coli BL21 (DE3).
+'''Fig.1 Schematic diagram of the overall design of quorum sensing system'''
-figure(plasmids+circuit)
+In order to add the quorum sensing system to the detection system, we set up the following gene circuit and double transferred the two plasmids into <i>E. coli BL21 (DE3)</i>(Fig.2).
-We selected the single colony overnight shaking bacteria carrying two plasmids from the medium plate containing two kinds of resistance, diluted the bacterial solution to OD = 2, and the final volume was 5ml. Then, when the bacterial solution grows to OD = 0.4 ~ 0.6, IPTG is added to induce the expression of pmrb and PmrA, and S protein is added. After PmrB feels the stimulation of S protein, PmrC is activated through a series of reactions to start the expression of LuxI and LuxR downstream. LuxI begins to continuously synthesize AHL signal molecules and enter the outside of the cell. After reaching a certain threshold, it can enter other engineering bacteria, Combined with endogenous LuxR to form a complex, activate the corresponding promoter plux_HS and further express the downstream reporter gene EGFP.
+https://static.igem.org/mediawiki/parts/6/68/T--NEU_CHINA--suoxiaoban.png
-We set up different experimental groups, and the results are as follows.
+'''Fig.2 The two plasmids were constructed with pETDuet-1 and pACYCDuet-1 as the vectors, and were co-transformed to <i>E.coli BL21(DE3)</i> to verify the quorum sensing system'''
-figure(results)
+Overnight shaking bacteria carrying two plasmids from the medium plate containing two kinds of resistance, diluted the bacterial solution to OD = 2, and the final volume was 5ml. Then, when the bacterial solution grows to OD = 0.4 ~ 0.6, IPTG is added to induce the expression of PmrB and PmrA, and S protein is added. After PmrB feels the stimulation of S protein, PmrC is activated through a series of reactions to start the expression of LuxI and LuxR downstream. LuxI begins to continuously synthesize AHL signal molecules and enter the outside of the cell. After reaching a certain threshold, it can enter other engineering bacteria, Combined with endogenous LuxR to form a complex, activate the corresponding promoter Plux_HS and further express the downstream reporter gene EGFP.
+We set up different experimental groups, and the results are as follows(Fig.3).
+https://static.igem.org/mediawiki/parts/1/1c/T--NEU_CHINA--dpp4_qssuoxiaoban.png
+'''Fig.3 The fluorescence intensity of engineered bacteria without quorum sensing system and with quorum sensing'''
 By joining the quorum sensing system. When a few engineering bacteria detect the virus, the information can be transmitted to other engineering bacteria through the mediation of signal molecule AHL, and finally the population expresses the reporter gene EGFP, which makes the detection results significant.