Difference between revisions of "Part:BBa K3487010"

Revision as of 11:10, 20 October 2020

A kill switch regulated by lysine concentration

Description

RS is a riboswitch regulated by lysine, which regulates the concentration of lysine to turn on or off downstream gene expression;RalR is a toxic protein that can cleave methylated and unmethylated DNA, leading to cell death.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 12
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 284
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 256

Result

We have constructed a suicide mechanism to ensure that the transgenic bacteria will not be harmful to the environment. The results of the construction of the recombinant plasmid pET28a-RS-RalR are shown in the following.

We put the lysine riboswitch connected sfGFP engineered bacteria in different concentrations of lysine for 24 hours, and then tested the fluorescent expression of the bacteria. The results are shown in the following.

The results show that the bacteria are in the lysine at a concentration of 30μmol/mL. Fluorescence expression was significantly inhibited, indicating that the lysine riboswitch can work normally.

We streaked the engineered strain into a 1Mm IPTG plate for overnight culture, and observed that the engineered strain containing the RalR gene did not grow on the plate. The expression of RalR was good under the positive and negative controls, and the colony situation in the corresponding area was significantly less than that of the empty vector control group. The result is shown in the following.

File:T--SZPT-China--RalR2.png

Reference

Guo Y, Quiroga C, Chen Q, et al. RalR (a DNase) and RalA (a small RNA) form a type I toxin-antitoxin system in Escherichia coli. Nucleic Acids Res. 2014;42(10):6448-6462.
Garst AD, Héroux A, Rambo RP, Batey RT. Crystal structure of the lysine riboswitch regulatory mRNA element. J Biol Chem. 2008;283(33):22347-22351.

@@ Line 16: / Line 16: @@
 ===Result===
 We have constructed a suicide mechanism to ensure that the transgenic bacteria will not be harmful to the environment. The results of the construction of the recombinant plasmid pET28a-RS-RalR are shown in the following.
-[[File:T--SZPT-CHINA--RS-RalR.png|thumb|center|500px|1% agarose gel image showing the results of PCR and double digestion
+[[File:T--SZPT-CHINA--RS-RalR.png|center|500px|]]
-M,D2000 Plus Marker;
-Line 1~3, pET28a（+）-RS-RalR double restriction digestion verification;
+We put the lysine riboswitch connected sfGFP engineered bacteria in different concentrations of lysine for 24 hours, and then tested the fluorescent expression of the bacteria. The results are shown in the following.
-Line 4~6，RS-RalR PCR verification]]
+[[File:T--SZPT-CHINA--RS3.png|700px|center]]
+The results show that the bacteria are in the lysine at a concentration of 30μmol/mL. Fluorescence expression was significantly inhibited, indicating that the lysine riboswitch can work normally.
+We streaked the engineered strain into a 1Mm IPTG plate for overnight culture, and observed that the engineered strain containing the RalR gene did not grow on the plate.
+The expression of RalR was good under the positive and negative controls, and the colony situation in the corresponding area was significantly less than that of the empty vector control group. The result is shown in the following.
+[[File:T--SZPT-China--RalR2.png|center|500px|]]
 ===Reference===