Difference between revisions of "Part:BBa K855008"

Revision as of 09:49, 20 October 2021

AHL-inducible Production of pvdQ (K855005)

This part requires the external presence acyl homoserine lactones to stimulate the production of PVDQ, but PVDQ produced will degrade the AHL molecules, it is a dynamic system that maintains the AHL concentration at certain level.

Characterization by BS_United_China 2021

See detail in part BBa_K3882002 PVDQ has been known by us and the University of Hong Kong in 2012 as the degrading enzyme of AHL (the most important quorum sensing molecule of Gram-negative bacteria). But in the part we designed this year, we connected the PVDQ protein and eGFP protein with three HA protein linkers, and added two pairs of 6 his tags. Our design is more intuitive and easier to extract PVDQ protein. And we also used the T7 promoter to increase protein expression.

Our ultimate goal in designing this part is to apply the PVDQ protein to daily life. We also designed a plasmid that can detect the concentration of AHL in food. See details in BBa_K3882001 After connecting the two parts we designed, we can detect the growth of bacteria in fresh food and secrete PVDQ protein in time to prevent the flora from releasing toxic substances.

The experiments conducted by our team in the laboratory in 2021 proved: 1. PVDQ protein can inhibit the growth of bacteria (Pseudomonas aeruginosa)

File:OD vaue of P.aeruginosa with PVDQ.png

Figure1:OD value of 4 bottles of 200ml Pseudomonas aeruginosa liquid cultivate at a constant temperature of 37 degrees(measured every hour) Control Group:Normal BL21 Escherichia coli E1: add 45ul 0.245mg/ml PVDQ extract before inoculation E5: 45ul 0.034mg/ml E8: 0.004 mg/ml

It has been speculated that the weakening of PVDQ inhibitory effect after 9 hours is due to protein denaturation, decomposition and inactivation under a constant temperature culture environment of 37 degrees Celsius.

2. PVDQ-eGFP was successfully extracted by our non-denaturing elution method and can be stored at -40 degrees Celsius without inactivation.

The PVDQ-eGFP produced by BBa_K3882002 induced by IPTG was observed under a fluorescence microscope

3. IPTG can activate the T7 promoter to enhance the expression of its downstream genes
4. The growth of BL21 E. coli inserted into our plasmid is not significantly different from that of normal E. coli
5. PVDQ-eGFP has not been decomposed after 30 minutes of treatment in a simulated gastric acid environment

Figure3: Add PVDQ to a mixed solution of 3% pH=0.61 hydrochloric acid and 17% pH=2.14 acetic acid at 37 degrees Celsius to decompose and react for half an hour. The picture shows the result of SDS-PAGE protein gel electrophoresis after the reaction

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 2284
Illegal XhoI site found at 2682
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 1119
Illegal NgoMIV site found at 1858
Illegal NgoMIV site found at 2260
Illegal NgoMIV site found at 2417
Illegal NgoMIV site found at 2644
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1005
Illegal SapI.rc site found at 3014

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K855008 short</partinfo>
-This part requires the external presence acyl homoserine lactones to stimulate the production of pvdQ, but pvdQ produced will degrade the AHL molecules, it is a dynamic system that maintains the AHL concentration at certain level.
+This part requires the external presence acyl homoserine lactones to stimulate the production of PVDQ, but PVDQ produced will degrade the AHL molecules, it is a dynamic system that maintains the AHL concentration at certain level.
 ==<b>Characterization by BS_United_China 2021</b>==
 See detail in part <partinfo>BBa_K3882002</partinfo>
-pvdQ has been known by us and the University of Hong Kong in 2012 as the degrading enzyme of AHL (the most important quorum sensing molecule of Gram-negative bacteria). But in the part we designed this year, we connected the pvdQ protein and eGFP protein with three HA protein linkers, and added two pairs of 6 his tags. Our design is more intuitive and easier to extract pvdQ protein. And we also used the T7 promoter to increase protein expression.<br>
+PVDQ has been known by us and the University of Hong Kong in 2012 as the degrading enzyme of AHL (the most important quorum sensing molecule of Gram-negative bacteria). But in the part we designed this year, we connected the PVDQ protein and eGFP protein with three HA protein linkers, and added two pairs of 6 his tags. Our design is more intuitive and easier to extract PVDQ protein. And we also used the T7 promoter to increase protein expression.<br>
 [[File:Part2 sequence.png|800px]]
-Our ultimate goal in designing this part is to apply the pvdQ protein to daily life. We also designed a plasmid that can detect the concentration of AHL in food.
+Our ultimate goal in designing this part is to apply the PVDQ protein to daily life. We also designed a plasmid that can detect the concentration of AHL in food.
 See details in <partinfo>BBa_K3882001</partinfo>
-After connecting the two parts we designed, we can detect the growth of bacteria in fresh food and secrete pvdQ protein in time to prevent the flora from releasing toxic substances.<br>
+After connecting the two parts we designed, we can detect the growth of bacteria in fresh food and secrete PVDQ protein in time to prevent the flora from releasing toxic substances.<br>
 The experiments conducted by our team in the laboratory in 2021 proved:
-. pvdQ protein can inhibit the growth of bacteria (Pseudomonas aeruginosa)<br>
+. PVDQ protein can inhibit the growth of bacteria (Pseudomonas aeruginosa)<br>
-[[File:OD vaue of P.aeruginosa with pvdQ.png|800px|thumb|center|<b> Figure1:OD value of 4 bottles of 200ml Pseudomonas aeruginosa liquid cultivate at a constant temperature of 37 degrees(measured every hour)</b> Control Group:Normal BL21 Escherichia coli E1: add 45ul 0.245mg/ml pvdQ extract before inoculation E5: 45ul 0.034mg/ml E8: 0.004 mg/ml ]]<br>
+[[File:OD vaue of P.aeruginosa with PVDQ.png|800px|thumb|center|<b> Figure1:OD value of 4 bottles of 200ml Pseudomonas aeruginosa liquid cultivate at a constant temperature of 37 degrees(measured every hour)</b> Control Group:Normal BL21 Escherichia coli E1: add 45ul 0.245mg/ml PVDQ extract before inoculation E5: 45ul 0.034mg/ml E8: 0.004 mg/ml ]]<br>
-It has been speculated that the weakening of pvdQ inhibitory effect after 9 hours is due to protein denaturation, decomposition and inactivation under a constant temperature culture environment of 37 degrees Celsius.
+It has been speculated that the weakening of PVDQ inhibitory effect after 9 hours is due to protein denaturation, decomposition and inactivation under a constant temperature culture environment of 37 degrees Celsius.
-. pvdQ-eGFP was successfully extracted by our non-denaturing elution method and can be stored at -40 degrees Celsius without inactivation.<br>
+. PVDQ-eGFP was successfully extracted by our non-denaturing elution method and can be stored at -40 degrees Celsius without inactivation.<br>
-[[File:1+IPTG.tif|780px|thumb|center|<b>The pvdQ-eGFP produced by <partinfo>BBa_K3882002</partinfo> induced by IPTG was observed under a fluorescence microscope </b>]]<br>
+[[File:1+IPTG.tif|780px|thumb|center|<b>The PVDQ-eGFP produced by <partinfo>BBa_K3882002</partinfo> induced by IPTG was observed under a fluorescence microscope </b>]]<br>
 . IPTG can activate the T7 promoter to enhance the expression of its downstream genes<br>
 . The growth of BL21 E. coli inserted into our plasmid is not significantly different from that of normal E. coli<br>
-. pvdQ-eGFP has not been decomposed after 30 minutes of treatment in a simulated gastric acid environment
+. PVDQ-eGFP has not been decomposed after 30 minutes of treatment in a simulated gastric acid environment
-[[File:PvdQ after 30 minutes acid environment.jpg|600px|thumb|center|<b> Figure3: Add pvdQ to a mixed solution of 3% pH=0.61 hydrochloric acid and 17% pH=2.14 acetic acid at 37 degrees Celsius to decompose and react for half an hour. The picture shows the result of SDS-PAGE protein gel electrophoresis after the reaction </b>]]
+[[File:PvdQ after 30 minutes acid environment.jpg|600px|thumb|center|<b> Figure3: Add PVDQ to a mixed solution of 3% pH=0.61 hydrochloric acid and 17% pH=2.14 acetic acid at 37 degrees Celsius to decompose and react for half an hour. The picture shows the result of SDS-PAGE protein gel electrophoresis after the reaction </b>]]
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