Difference between revisions of "Part:BBa K4645014"

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<partinfo>BBa_K4645014 short</partinfo>
 
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RelE is a toxin protein that inhibits translation. RNA thermosensors which could inhibit downstream gene expression when the temperature is 37 &#8451; but not affect downstream gene expression significantly when the temperature is below 28 &#8451;.The combination of two components can regulate the expression of toxin proteins by changing the temperature, and then control the death of engineering bacteria.
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RelE is a toxin protein that inhibits translation. RNA thermosensors which could inhibit downstream gene expression when the temperature is 37 &#8451; but not affect downstream gene expression significantly when the temperature is below 28 &#8451;. The combination of two components can regulate the expression of toxin proteins by changing the temperature, and then control the death of engineering bacteria.
 
The circuit is shown in the figure below.
 
The circuit is shown in the figure below.
 
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<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/rnarele.png" style="width:50%; "></center>
 
<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/rnarele.png" style="width:50%; "></center>
 
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<center><b>Figure 1. The circuit of RNA thermosensors and RelE.</b> </center>
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==Basic Elements==
 
==Basic Elements==
<p><b>RNA thermosensors :</b>This is one of heat-repressible RNA thermosensors which could inhibit downstream gene expression when the temperature is 37 &#8451; but not affect downstream gene expression significantly when the temperature is below 26 &#8451;.
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<p><b>RNA thermosensors:</b> This is one of heat-repressible RNA thermosensors which could inhibit downstream gene expression when the temperature is 37 &#8451; but not affect downstream gene expression significantly when the temperature is below 26 &#8451;.
 
</p>
 
</p>
<p><b>RelE toxin :</b>The RelE toxin is an RNase that preferentially cleaves mRNAs bound to the ribosome at the second position of stop codons. </p>
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<p><b>RelE toxin:</b> The RelE toxin is an RNase that preferentially cleaves mRNAs bound to the ribosome at the second position of stop codons. </p>
  
 
==Basic Elements Functional validation ==
 
==Basic Elements Functional validation ==
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====Test Design (Method 1)====
 
====Test Design (Method 1)====
First we placed the Amcyan protein after the RNA thermosensor in the PUC57 plasmid and expressed the plasmid in E.coli DN5α. After culturing for 4 hours continuously at 36°C, 26°C and 16°C respectively, the fluorescence intensity was detected and divided by the OD600 value, with DH5α cultured at 36°C for 4 hours without plasmid transformation as control. The relative fluorescence intensity was detected to verify the function of the RNA thermosensor.
+
First we placed the AmCyan protein after the RNA thermosensor in the PUC57 plasmid and expressed the plasmid in <i>E. coli</i> DH5α. After culturing for 4 hours continuously at 36℃, 26℃ and 16℃ respectively, the fluorescence intensity was detected and divided by the OD<sub>600</sub> value, with DH5α cultured at 36℃ for 4 hours without plasmid transformation as control. The relative fluorescence intensity was detected to verify the function of the RNA thermosensor.
 
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====Test Protocol (Method 1)====
 
====Test Protocol (Method 1)====
<p>&nbsp;&nbsp;&nbsp;&nbsp;1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli DH5α. Then spread it onto LB medium plates with 170 μg/mL chloramphenicol and incubate overnight at 37 °C in an incubator. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;1) Methods of molecular cloning and transformation are described above. Transform this plasmid into <i>E. coli</i> DH5α. Then spread it onto LB medium plates with 170 μg/mL chloramphenicol and incubate overnight at 37 in an incubator. </p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;2)  Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 170 μg/mL chloramphenicol and cultured at temperature (36 °C) respectively while shaking at 200 rpm.</p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;2)  Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 170 μg/mL chloramphenicol and cultured at temperature (36 ) respectively while shaking at 200 rpm.</p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;3) Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader) until the OD600 of the bacteria solution reaches 0.4-0.6. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;3) Measure the OD<sub>600</sub> value of the resuspension culture media in an automatic microplate reader (<b>Synergy</b><sup>TM</sup> H1 hybrid multimodal reader) until the OD<sub>600</sub> of the bacteria solution reaches 0.4-0.6. </p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;4) After grouping the samples, culture them continuously for 4 hours at 36 °C, 26 °C and 16 °C respectively while shaking at 200 rpm.</p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;4) After grouping the samples, culture them continuously for 4 hours at 36℃, 26℃ and 16℃ respectively while shaking at 200 rpm.</p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;5) Continuously measure fluorescence intensity (Exλ:453nm Emλ:486nm) and OD600 using an automatic microplate reader (SynergyH1 hybrid multimodal reader). After detection, divide the measured fluorescence intensity by OD600 to obtain the relative fluorescence intensity and analyze the results.</p>
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<p>&nbsp;&nbsp;&nbsp;&nbsp;5) Continuously measure fluorescence intensity (Exλ:453nm Emλ:486nm) and OD<sub>600</sub> using an automatic microplate reader (<b>Synergy</b><sup>TM</sup> H1 hybrid multimodal reader). After detection, divide the measured fluorescence intensity by OD<sub>600</sub> to obtain the relative fluorescence intensity and analyze the results.</p>
  
 
====Result(Method 1)====
 
====Result(Method 1)====
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<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/rna2-1.jpg" style="width:60%; "></center>
 
<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/rna2-1.jpg" style="width:60%; "></center>
 
<br>
 
<br>
<center><b>Figure 1.Analysis of Differential Relative Fluorescence Intensity under Four Hours of Different Temperature Induction Conditions </b> </center>
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<center><b>Figure 1. Analysis of Differential Relative Fluorescence Intensity under Four Hours of Different Temperature Induction Conditions </b> </center>
 
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====Test Design (Method 2)====
 
====Test Design (Method 2)====
We referred to the verification method of<b> iGEM21_HZAU-China BBa_K3733043</b>to verify the RNA thermosensor. We connected the toxin protein HepT after the RNA thermosensor and expressed it in E.coli DN5α using a plasmid.  
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We referred to the verification method of<b> iGEM21_HZAU-China BBa_K3733043</b> to verify the RNA thermosensor. We connected the toxin protein HepT after the RNA thermosensor and expressed it in <i>E. coli</i> DH5α using a plasmid.  
 
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====Test Protocol (Method 2)====
 
====Test Protocol (Method 2)====
<p>&nbsp;&nbsp;&nbsp;&nbsp;1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli DH5α. Then spread it onto LB medium plates with 170 μg/mL chloramphenicol and incubate overnight at 37 °C in an incubator.</p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;1) Methods of molecular cloning and transformation are described above. Transform this plasmid into <i>E. coli</i> DH5α. Then spread it onto LB medium plates with 170 μg/mL chloramphenicol and incubate overnight at 37 °C in an incubator.</p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 170 μg/mL chloramphenicol and cultured at temperature (37 °C) respectively while shaking at 200 rpm overnight.</p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 170 μg/mL chloramphenicol and cultured at temperature (37℃) respectively while shaking at 200 rpm overnight.</p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;3) Measure the OD600 value of the resuspending culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader) until the OD600 of the bacteria solution reaches 0.4-0.6. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;3) Measure the OD<sub>600</sub> value of the resuspending culture media in an automatic microplate reader (<b>Synergy</b><sup>TM</sup> H1 hybrid multimodal reader) until the OD<sub>600</sub> of the bacteria solution reaches 0.4-0.6. </p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;4) After grouping the samples, culture them continuously for 4 hours at 36°C and 26°C respectively, while shaking at 200 rpm.</p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;4) After grouping the samples, culture them continuously for 4 hours at 36℃ and 26℃ respectively, while shaking at 200 rpm.</p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;5) Continuously measure fluorescence intensity (Exλ:453nm Emλ:486nm) and OD600 using an automatic microplate reader (SynergyH1 hybrid multimodal reader). After detection, divide the measured fluorescence intensity by OD600 to obtain the relative fluorescence intensity and analyze the results.</p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;5) Continuously measure fluorescence intensity (Exλ:453nm Emλ:486nm) and OD<sub>600</sub> using an automatic microplate reader (<b>Synergy</b><sup>TM</sup> H1 hybrid multimodal reader). After detection, divide the measured fluorescence intensity by OD<sub>600</sub> to obtain the relative fluorescence intensity and analyze the results.</p>
  
 
====Result(Method 2)====
 
====Result(Method 2)====
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<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/rna2-2.jpg" style="width:60%; "></center>
 
<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/rna2-2.jpg" style="width:60%; "></center>
 
<br>
 
<br>
<center><b>Figure 2.Bacterial OD600 over time </b></center>
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<center><b>Figure 2. Bacterial OD<sub>600</sub> over time </b></center>
 
<br>
 
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<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/img-20231007-152022.jpg" style="width:50%; "></center>
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<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/img-20231007-1520221.jpg" style="width:50%; "></center>
 
<br>
 
<br>
<center><b>Figure 3.Phenotypic result comparison chart </b></center>
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<center><b>Figure 3. Phenotypic result comparison chart </b></center>
 
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====Analysis of the experimental results====
 
====Analysis of the experimental results====
 
Results and analysis:
 
Results and analysis:
Both verification methods successfully validated that the RNA thermosensor can function and express proteins at 26°C or lower temperatures. However, the experimental results also showed that there was some leakage from the RNA thermosensor at 36°C, which may lead to some adverse effects.
+
Both verification methods successfully validated that the RNA thermosensor can function and express proteins at 26℃ or lower temperatures. However, the experimental results also showed that there was some leakage from the RNA thermosensor at 36℃, which may lead to some adverse effects.
  
 
====Model====
 
====Model====
In order to gain a clearer understanding and validate the feasibility of the RNA thermosensor, we used DINAMelt's server for analysis of the neck ring structure.In the experiment, we obtained the fluorescence intensity of three points and assigned high weights to the experimental values, which in turn transformed into the probability of opening the loop. We have obtained the following graph <b>Figure 4.</b>, which, although different from the open-loop probability graph we recognize, does indeed match the results of our experiment.
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In order to gain a clearer understanding and validate the feasibility of the RNA thermosensor, we used DINAMelt's server for analysis of the neck ring structure.In the experiment, we obtained the fluorescence intensity of three points and assigned high weights to the experimental values, which in turn transformed into the probability of opening the loop. We have obtained the following graph <b>Figure 4</b>, which, although different from the open-loop probability graph we recognize, does indeed match the results of our experiment.
 
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<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/image.png" style="width:50%; "></center>
 
<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/image.png" style="width:50%; "></center>
 
<br>
 
<br>
<center><b>Figure 4.RNA thermosensor open-loop probability chart </b></center>
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<center><b>Figure 4. RNA thermosensor open-loop probability chart. </b></center>
 
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</body>
 
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</html>
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===RelE toxin===
 
===RelE toxin===
 
<b>To validate the function of the RelE toxin, we used two methods to experimentally verify it.</b> 
 
<b>To validate the function of the RelE toxin, we used two methods to experimentally verify it.</b> 
 
====Test Design====
 
====Test Design====
In our experiment, we constructed a plasmid vector with RelE toxin protein connected after the lactose promoter and introduced it into E.coli BL21(DE3). IPTG was used to induce expression and OD600 was detected continuously for 4 hours. Samples were taken every 30 minutes, diluted and plated to determine viable cell counts. 
+
In our experiment, we constructed a plasmid vector with RelE toxin protein connected after the lactose promoter and introduced it into <i>E. coli</i> BL21(DE3). IPTG was used to induce expression and OD<sub>600</sub> was detected continuously for 4 hours. Samples were taken every 30 minutes, diluted and plated to determine viable cell counts. 
 +
 
 
====Test Protocol====
 
====Test Protocol====
<b>Toxin protein RelE OD600 detection</b>
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<b>Toxin protein RelE OD<sub>600</sub> detection</b>
<p>&nbsp;&nbsp;&nbsp;&nbsp;1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli BL21. Then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37 °C in an incubator. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;1) Methods of molecular cloning and transformation are described above. Transform this plasmid into <i>E. coli</i> BL21. Then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37℃ in an incubator. </p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 50 μg/mL kanamycin and cultured at temperature (37 °C) respectively while shaking at 200 rpm. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 50 μg/mL kanamycin and cultured at temperature (37℃) respectively while shaking at 200 rpm. </p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;3) Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader) until the OD600 of the bacteria solution reaches 0.4-0.6. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;3) Measure the OD<sub>600</sub> value of the resuspension culture media in an automatic microplate reader (<b>Synergy</b><sup>TM</sup> H1 hybrid multimodal reader) until the OD<sub>600</sub> of the bacteria solution reaches 0.4-0.6. </p>
 
<p>&nbsp;&nbsp;&nbsp;&nbsp;4)  Add IPTG to the bacteria solution of the experimental group to induce expression of the toxin proteins.</p>
 
<p>&nbsp;&nbsp;&nbsp;&nbsp;4)  Add IPTG to the bacteria solution of the experimental group to induce expression of the toxin proteins.</p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;5) Plot the OD600 value curves of the resuspension culture media over time in an automatic microplate reader (SynergyH1 hybrid multimodal reader). Incubate the cultures for 4 hours at 37°C while shaking at 200 rpm. Take samples at intervals or continuously measure the OD600 data of the bacteria solution with a UV spectrophotometer. And then convert the raw data into OD600. Compare the data of experimental groups and control group and compare curves in two schemes with each other.</p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;5) Plot the OD<sub>600</sub> value curves of the resuspension culture media over time in an automatic microplate reader (<b>Synergy</b><sup>TM</sup> H1 hybrid multimodal reader). Incubate the cultures for 4 hours at 37℃ while shaking at 200 rpm. Take samples at intervals or continuously measure the OD<sub>600</sub> data of the bacteria solution with a UV spectrophotometer. And then convert the raw data into OD<sub>600</sub>. Compare the data of experimental groups and control group and compare curves in two schemes with each other.</p>
  
 
<b>Toxin protein RelE CFU detection</b>
 
<b>Toxin protein RelE CFU detection</b>
<p>&nbsp;&nbsp;&nbsp;&nbsp;1) 1. Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli BL21. Then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37 °C in an incubator.</p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;1) 1. Methods of molecular cloning and transformation are described above. Transform this plasmid into <i>E. coli</i> BL21(DE3). Then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37℃ in an incubator.</p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 50 μg/mL kanamycin and cultured at temperature (37 °C) respectively while shaking at 200 rpm.</p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 50 μg/mL kanamycin and cultured at temperature (37℃) respectively while shaking at 200 rpm.</p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;3) Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader) until the OD600 of the bacteria solution reaches 0.4-0.6. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;3) Measure the OD<sub>600</sub> value of the resuspension culture media in an automatic microplate reader (<b>Synergy</b><sup>TM</sup> H1 hybrid multimodal reader) until the OD<sub>600</sub> of the bacteria solution reaches 0.4-0.6. </p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;4) Take samples every half hour, dilute the bacteria solution 1*106times, then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37 °C in an incubator.</p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;4) Take samples every half hour, dilute the bacteria solution 1*106 times, then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37℃ in an incubator.</p>
 
<p>&nbsp;&nbsp;&nbsp;&nbsp;5)Count colonies on the plates cultured for the same time the next day, process the statistical data and plot the image.</p>
 
<p>&nbsp;&nbsp;&nbsp;&nbsp;5)Count colonies on the plates cultured for the same time the next day, process the statistical data and plot the image.</p>
  
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<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/rele2-1.jpg" style="width:50%; "></center>
 
<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/rele2-1.jpg" style="width:50%; "></center>
 
<br>
 
<br>
<center><b>Figure 1. Bacterial OD600 over time .</b> </center>
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<center><b>Figure 5. Bacterial OD<sub>600</sub> over time .</b> </center>
 
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<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/rele2-2-ab.jpg" style="width:80%; "></center>
 
<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/rele2-2-ab.jpg" style="width:80%; "></center>
 
<br>
 
<br>
<center><b>Figure 2. Viable cell counts over time.</b></center>
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<center><b>Figure 6. Viable cell counts over time.</b></center>
 
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====Analysis of the experimental results====
 
====Analysis of the experimental results====
Since RelE is a weak toxin protein, as shown in Figure 1., the OD600 detection showed obvious inhibition of bacterial growth after induced expression, which delayed the log phase, but the bacteria could still grow in the later phase. The viable cell count method also produced significant results. As shown in Figure 2., viable cell counts decreased rapidly within one hour after induction, exhibiting significant growth inhibition, which demonstrated the bacteriostatic effect of this toxin. 
+
Since RelE is a weak toxin protein, as shown in <b>Figure 5</b>, the OD<sub>600</sub> detection showed obvious inhibition of bacterial growth after induced expression, which delayed the log phase, but the bacteria could still grow in the later phase. The viable cell count method also produced significant results. As shown in <b>Figure 6</b>, viable cell counts decreased rapidly within one hour after induction, exhibiting significant growth inhibition, which demonstrated the bacteriostatic effect of this toxin. 
 +
 
 
==Composite Elements Functional validation ==
 
==Composite Elements Functional validation ==
 
===Combination element of RNA thermosensors regulatory RelE toxin protein===
 
===Combination element of RNA thermosensors regulatory RelE toxin protein===
 
<b>The combination of two components can regulate the expression of toxin proteins by changing the temperature, and then control the death of engineering bacteria.</b>
 
<b>The combination of two components can regulate the expression of toxin proteins by changing the temperature, and then control the death of engineering bacteria.</b>
 
====Test Design ====
 
====Test Design ====
we placed the RelE protein after the RNA thermosensor in the PUC57 plasmid and expressed the plasmid in E.coli BL21(DE3).Both control and treated groups were incubated at 37°C for 2.5 hrs and at 27°C for 4.5 hrs, and the values of OD 600 were measured.
+
we placed the RelE protein after the RNA thermosensor in the PUC57 plasmid and expressed the plasmid in <i>E. coli</i> BL21(DE3). Both control and treated groups were incubated at 37°C for 2.5 h and at 27℃ for 4.5 h, and the values of OD<sub>600</sub> were measured.
 
====Test Protocol====
 
====Test Protocol====
<p>&nbsp;&nbsp;&nbsp;&nbsp;1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E.coli BL21(DE3). Then spread it onto LB medium plates with 170 μg/mL chloramphenicol and incubate overnight at 37 °C in an incubator. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;1) Methods of molecular cloning and transformation are described above. Transform this plasmid into <i>E. coli</i> BL21(DE3). Then spread it onto LB medium plates with 170 μg/mL chloramphenicol and incubate overnight at 37℃ in an incubator. </p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 170 μg/mL chloramphenicol and cultured at temperature (36 °C) respectively while shaking at 200 rpm. </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 170 μg/mL chloramphenicol and cultured at temperature (36℃) respectively while shaking at 200 rpm. </p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;3) Firstly,Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader)for 2.5 hours at temperature (37 °C),then Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader)for 4.5 hours at temperature (27 °C) </p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;3) Firstly,Measure the OD<sub>600</sub> value of the resuspension culture media in an automatic microplate reader (<b>Synergy</b><sup>TM</sup> H1 hybrid multimodal reader)for 2.5 hours at temperature (37℃),then Measure the OD<sub>600</sub> value of the resuspension culture media in an automatic microplate reader (<b>Synergy</b><sup>TM</sup> H1 hybrid multimodal reader) for 4.5 hours at temperature (27℃) </p>
<p>&nbsp;&nbsp;&nbsp;&nbsp;4) Take samples at intervals or continuously measure the OD600 data of the bacteria solution with a UV spectrophotometer. And then convert the raw data into OD600. Compare the data of experimental groups and control group and compare curves in two schemes with each other.</p>
+
<p>&nbsp;&nbsp;&nbsp;&nbsp;4) Take samples at intervals or continuously measure the OD<sub>600</sub> data of the bacteria solution with a UV spectrophotometer. And then convert the raw data into OD<sub>600</sub>. Compare the data of experimental groups and control group and compare curves in two schemes with each other.</p>
 +
 
 
====Result====
 
====Result====
 
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<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/releax.jpg" style="width:50%; "></center>
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<center><img src="https://static.igem.wiki/teams/4645/wiki/wet-lab/suicide/releax2.jpg" style="width:50%; "></center>
 
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<br>
<center><b>Figure 1.The control and Treated groups were now incubated at 37°C for 2.5 hours and then placed at 27°C for 4.5 hours.</b> </center>
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<center><b>Figure 7. The control and Treated groups were now incubated at 37℃ for 2.5 hours and then placed at 27℃ for 4.5 hours.</b> </center>
 
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====Analysis of the experimental results====
 +
Changes in temperature can induce the expression of RelE toxin proteins as shown in <b>Figure 7</b>, the OD<sub>600</sub> detection showed obvious inhibition of bacterial growth after changed temperature, which delayed the log phase, but the bacteria could still grow in the later phase.At the same time the temperature also has some slight effect on the growth of the bacteria.

Latest revision as of 15:57, 12 October 2023


Combination element of RNA thermosensors regulatory RelE toxin protein

RelE is a toxin protein that inhibits translation. RNA thermosensors which could inhibit downstream gene expression when the temperature is 37 ℃ but not affect downstream gene expression significantly when the temperature is below 28 ℃. The combination of two components can regulate the expression of toxin proteins by changing the temperature, and then control the death of engineering bacteria. The circuit is shown in the figure below. 无标题文档


Basic Elements

RNA thermosensors: This is one of heat-repressible RNA thermosensors which could inhibit downstream gene expression when the temperature is 37 ℃ but not affect downstream gene expression significantly when the temperature is below 26 ℃.

RelE toxin: The RelE toxin is an RNase that preferentially cleaves mRNAs bound to the ribosome at the second position of stop codons.

Basic Elements Functional validation

RNA thermosensors

To validate the function of the RNA thermosensor, we used two methods to experimentally verify it. 

Test Design (Method 1)

First we placed the AmCyan protein after the RNA thermosensor in the PUC57 plasmid and expressed the plasmid in E. coli DH5α. After culturing for 4 hours continuously at 36℃, 26℃ and 16℃ respectively, the fluorescence intensity was detected and divided by the OD600 value, with DH5α cultured at 36℃ for 4 hours without plasmid transformation as control. The relative fluorescence intensity was detected to verify the function of the RNA thermosensor. 无标题文档


Test Protocol (Method 1)

    1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli DH5α. Then spread it onto LB medium plates with 170 μg/mL chloramphenicol and incubate overnight at 37 ℃ in an incubator.

    2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 170 μg/mL chloramphenicol and cultured at temperature (36 ℃) respectively while shaking at 200 rpm.

    3) Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyTM H1 hybrid multimodal reader) until the OD600 of the bacteria solution reaches 0.4-0.6.

    4) After grouping the samples, culture them continuously for 4 hours at 36℃, 26℃ and 16℃ respectively while shaking at 200 rpm.

    5) Continuously measure fluorescence intensity (Exλ:453nm Emλ:486nm) and OD600 using an automatic microplate reader (SynergyTM H1 hybrid multimodal reader). After detection, divide the measured fluorescence intensity by OD600 to obtain the relative fluorescence intensity and analyze the results.

Result(Method 1)

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Figure 1. Analysis of Differential Relative Fluorescence Intensity under Four Hours of Different Temperature Induction Conditions

Test Design (Method 2)

We referred to the verification method of iGEM21_HZAU-China BBa_K3733043 to verify the RNA thermosensor. We connected the toxin protein HepT after the RNA thermosensor and expressed it in E. coli DH5α using a plasmid. 无标题文档


Test Protocol (Method 2)

    1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli DH5α. Then spread it onto LB medium plates with 170 μg/mL chloramphenicol and incubate overnight at 37 °C in an incubator.

    2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 170 μg/mL chloramphenicol and cultured at temperature (37℃) respectively while shaking at 200 rpm overnight.

    3) Measure the OD600 value of the resuspending culture media in an automatic microplate reader (SynergyTM H1 hybrid multimodal reader) until the OD600 of the bacteria solution reaches 0.4-0.6.

    4) After grouping the samples, culture them continuously for 4 hours at 36℃ and 26℃ respectively, while shaking at 200 rpm.

    5) Continuously measure fluorescence intensity (Exλ:453nm Emλ:486nm) and OD600 using an automatic microplate reader (SynergyTM H1 hybrid multimodal reader). After detection, divide the measured fluorescence intensity by OD600 to obtain the relative fluorescence intensity and analyze the results.

Result(Method 2)

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Figure 2. Bacterial OD600 over time


Figure 3. Phenotypic result comparison chart

Analysis of the experimental results

Results and analysis: Both verification methods successfully validated that the RNA thermosensor can function and express proteins at 26℃ or lower temperatures. However, the experimental results also showed that there was some leakage from the RNA thermosensor at 36℃, which may lead to some adverse effects.

Model

In order to gain a clearer understanding and validate the feasibility of the RNA thermosensor, we used DINAMelt's server for analysis of the neck ring structure.In the experiment, we obtained the fluorescence intensity of three points and assigned high weights to the experimental values, which in turn transformed into the probability of opening the loop. We have obtained the following graph Figure 4, which, although different from the open-loop probability graph we recognize, does indeed match the results of our experiment. 无标题文档


Figure 4. RNA thermosensor open-loop probability chart.

RelE toxin

To validate the function of the RelE toxin, we used two methods to experimentally verify it. 

Test Design

In our experiment, we constructed a plasmid vector with RelE toxin protein connected after the lactose promoter and introduced it into E. coli BL21(DE3). IPTG was used to induce expression and OD600 was detected continuously for 4 hours. Samples were taken every 30 minutes, diluted and plated to determine viable cell counts. 

Test Protocol

Toxin protein RelE OD600 detection

    1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli BL21. Then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37℃ in an incubator.

    2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 50 μg/mL kanamycin and cultured at temperature (37℃) respectively while shaking at 200 rpm.

    3) Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyTM H1 hybrid multimodal reader) until the OD600 of the bacteria solution reaches 0.4-0.6.

    4) Add IPTG to the bacteria solution of the experimental group to induce expression of the toxin proteins.

    5) Plot the OD600 value curves of the resuspension culture media over time in an automatic microplate reader (SynergyTM H1 hybrid multimodal reader). Incubate the cultures for 4 hours at 37℃ while shaking at 200 rpm. Take samples at intervals or continuously measure the OD600 data of the bacteria solution with a UV spectrophotometer. And then convert the raw data into OD600. Compare the data of experimental groups and control group and compare curves in two schemes with each other.

Toxin protein RelE CFU detection

    1) 1. Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli BL21(DE3). Then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37℃ in an incubator.

    2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 50 μg/mL kanamycin and cultured at temperature (37℃) respectively while shaking at 200 rpm.

    3) Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyTM H1 hybrid multimodal reader) until the OD600 of the bacteria solution reaches 0.4-0.6.

    4) Take samples every half hour, dilute the bacteria solution 1*106 times, then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37℃ in an incubator.

    5)Count colonies on the plates cultured for the same time the next day, process the statistical data and plot the image.

Result

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Figure 5. Bacterial OD600 over time .


Figure 6. Viable cell counts over time.

Analysis of the experimental results

Since RelE is a weak toxin protein, as shown in Figure 5, the OD600 detection showed obvious inhibition of bacterial growth after induced expression, which delayed the log phase, but the bacteria could still grow in the later phase. The viable cell count method also produced significant results. As shown in Figure 6, viable cell counts decreased rapidly within one hour after induction, exhibiting significant growth inhibition, which demonstrated the bacteriostatic effect of this toxin. 

Composite Elements Functional validation

Combination element of RNA thermosensors regulatory RelE toxin protein

The combination of two components can regulate the expression of toxin proteins by changing the temperature, and then control the death of engineering bacteria.

Test Design

we placed the RelE protein after the RNA thermosensor in the PUC57 plasmid and expressed the plasmid in E. coli BL21(DE3). Both control and treated groups were incubated at 37°C for 2.5 h and at 27℃ for 4.5 h, and the values of OD600 were measured.

Test Protocol

    1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli BL21(DE3). Then spread it onto LB medium plates with 170 μg/mL chloramphenicol and incubate overnight at 37℃ in an incubator.

    2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 170 μg/mL chloramphenicol and cultured at temperature (36℃) respectively while shaking at 200 rpm.

    3) Firstly,Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyTM H1 hybrid multimodal reader)for 2.5 hours at temperature (37℃),then Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyTM H1 hybrid multimodal reader) for 4.5 hours at temperature (27℃)

    4) Take samples at intervals or continuously measure the OD600 data of the bacteria solution with a UV spectrophotometer. And then convert the raw data into OD600. Compare the data of experimental groups and control group and compare curves in two schemes with each other.

Result

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Figure 7. The control and Treated groups were now incubated at 37℃ for 2.5 hours and then placed at 27℃ for 4.5 hours.

Analysis of the experimental results

Changes in temperature can induce the expression of RelE toxin proteins as shown in Figure 7, the OD600 detection showed obvious inhibition of bacterial growth after changed temperature, which delayed the log phase, but the bacteria could still grow in the later phase.At the same time the temperature also has some slight effect on the growth of the bacteria.