Difference between revisions of "Part:BBa K3698003"
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__NOTOC__<partinfo>BBa_K3698003 short</partinfo>A blue DegP expression loop that can be regulated by CpxR.<!-- Add more about the biology of this part here===Usage and Biology===<!-- --><span class='h3bb'>Sequence and Features</span><partinfo>BBa_K3698003 SequenceAndFeatures</partinfo><!-- Uncomment this to enable Functional Parameter display ===Functional Parameters===<partinfo>BBa_K3698003 parameters</partinfo><!-- --><h2> Introduction </h2>This part is a composite part of <partinfo>BBa_K3698002</partinfo>, <partinfo>BBa_K3698001</partinfo>, <partinfo>BBa_K3698004</partinfo> and <partinfo>BBa_K592009</partinfo>, As shown in Figure 1. The BBa_K3698002 is regulatory region of degP, it contains three CpxR sites, which can bound Phosphorylated DNA-binding transcriptional dual regulator CpxR activates transcription of degP. The BBa_K3698002 is degP gene which expresses periplasmic serine endoprotease. This protease is responsible for digesting abnormally folded proteins in E. coli in a high temperature environment to protect E. coli. There is also experimental evidence that DegP is beneficial to E. coli to resist acid stress. For the convenience of characterization, we connected the degP gene fused with chromoprotein after the rDegP. This competent part was inserted into a plasmid containing the chloramphenicol resistance gene for characterization, as shown in Figure 2.<html> <body> <img src="https://2020.igem.org/wiki/images/4/4a/T--XHD-ShanDong-China--Engineering-Figure7.jpeg" style="width:60%"> </body></html>Figure1. Schematic diagram of degP_CP circuit<html> <body> <img src="https://2020.igem.org/wiki/images/7/73/T--XHD-ShanDong-China--Engineering-Figure10.jpeg" style="width:60%"> </body></html>Figure2. Plasmid map of pRdegPLCP<h2> Experiment and Results </h2>We first constructed a composite fragment of rDegP and degP. The result is shown in Figure 3. The fragment size is 1757bp. Then the pre-constructed pamilCP was linearized by PCR. The result is shown in Figure 4, and the fragment size is 2876bp. The fragments recovered and purified in the first two steps are transformed into DH5α competent after homologous recombination, and spread on a chloramphenicol-resistant plate, and a single colony is picked for culture. The obtained culture fluid was used for PCR verification, and the primers used were Test-Top and Test-Bottom. After the PCR was completed, gel electrophoresis was performed. The result is shown in Figure 5. The target fragment should be 1000 bp, and the fragment obtained is in line with the expected size, confirming the successful recombination of our pRdegPLCP plasmid.<html> <body> <img src="https://2020.igem.org/wiki/images/c/c2/T--XHD-ShanDong-China--Engineering-Figure8.jpeg" style="width:60%"> </body></html>Figure3. Gel electrophoresis of rDegP and degP composite fragments<html> <body> <img src="https://2020.igem.org/wiki/images/8/87/T--XHD-ShanDong-China--Engineering-Figure9.jpeg" style="width:60%"> </body></html>Figure4. Gel electrophoresis of linearized pamilCP fragment<html> <body>  <img src="https://2020.igem.org/wiki/images/d/d6/T--XHD-ShanDong-China--Improvement-Figure5.jpeg" style="width:60%"> </body></html>Figure5. Gel electrophoresis of pRdegPLCP recombination verifiesThe pRdegPLCP was transformed into DH5α, and placed in normal temperature (37°C) and high temperature (45°C) respectively. The OD588 and OD600 of the bacterial solution were measured every 1h. After 11h, the DH5α_pRdegPLCP at the two temperatures was obtained. The growth curve, as shown in Figure 6, the curve in the high temperature environment is significantly lower than the normal temperature. After enriching the bacterial liquid, it can be seen that the bacterial liquid has a very obvious blue color, as shown in Figure 7. Divide OD588 by OD600 to obtain the absorbance of chromprotein amilCP to characterize the expression of degP, as shown in Figure 7. The expression level of degP at 45°C is obviously higher than that at 37°C. This shows that CpxR in our new part can normally regulate the expression of degP, and can respond to environmental changes.<html> <body> <img src="https://2020.igem.org/wiki/images/f/f0/T--XHD-ShanDong-China--Engineering-Figure11.jpeg" style="width:60%"> </body></html>Figure6. Growth curve of DH5α_pRdegPLCP<html> <body> <img src="https://2020.igem.org/wiki/images/9/99/T--XHD-ShanDong-China--Engineering-Figure12.jpeg" style="width:60%"> </body></html>Figure7. Enriched bacteria liquid<html> <body> <img src="https://2020.igem.org/wiki/images/f/ff/T--XHD-ShanDong-China--Engineering-Figure13.jpeg" style="width:60%"> </body></html>Figure8. Expression level of degP-amilCP
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<partinfo>BBa_K3698003 short</partinfo>
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A blue DegP expression loop that can be regulated by CpxR.
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3698003 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K3698003 parameters</partinfo>
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RdegPLCP
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<h2> Introduction </h2>
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The transcription of degP is regulated by CpxR. As shown in Figure 1, there are three CpxR sites upstream of degP. These three sites can bind to phosphorylated DNA and the transcription dual regulator CpxR to activate transcription. Our project will explore the relationship between the distance between the cpxR and degP genes expressing CpxR and the regulation efficiency, so these three CpxR sites and the degP gene will be amplified from the E. coli MG1655 genome by PCR. The primers obtained contained as shown in Figure 2, and the target fragment was 1757bp.
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<h2>Aim of experiment</h2>
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In order to characterize the expression level of degP, we fused a chromoprotein amilCP with it and connected it with a Linker to form a new element named degP_CP. This new element was inserted into the plasmid backbone containing CmR resistance to form the plasmid pdegP_CP. PCR verification showed that the pdegP_CP was successfully constructed. As shown in Figure 3, the target fragment was 1000 bp, and the resulting fragment was in line with expectations. The map of plasmid pRdegPLCP is shown in Figure 4. The plasmid contains a CmR resistance gene.
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Figure1. Schematic diagram of degP_CP circuit
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Figure2. Gel electrophoresis of degP and CpxR regulatory regions
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Figure3. Gel electrophoresis of pdegP_CP recombination verify.
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Figure4. Plasmid map of pdegP_CP
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<h2> Experiment and Results </h2>
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PdegP_CP was transformed into DH5α competent, and placed in normal temperature (37℃) and high temperature (45℃) respectively. The OD588 and OD600 of the bacterial solution were measured every 1h. After 12h, the DH5α_pdegP_CP at the two temperatures was obtained. The growth curve, as shown in Figure5, the curve in the high temperature environment is significantly lower than the normal temperature. After enriching the bacterial liquid, it can be seen that the bacterial liquid has a very obvious blue color, as shown in Figure 6. Divide OD588 by OD600 to obtain the absorbance of chromin to characterize the expression of degP, as shown in Figure 7，It can be clearly seen that the expression level of degP in a 45℃ environment is much higher than that in a 37℃ environment. This shows that CpxR in our new element can normally regulate the expression of degP and chromin, and can respond to environmental changes.
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Figure5. The growth curve of DH5α_pdegP_CP at two temperatures.
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Figure6. Photo of the fluid-rich.
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Figure7. Expression level of degP_CP at two temperatures
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Revision as of 18:30, 27 October 2020

RdegPLCPA blue DegP expression loop that can be regulated by CpxR.Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Introduction

This part is a composite part of BBa_K3698002, BBa_K3698001, BBa_K3698004 and BBa_K592009, As shown in Figure 1. The BBa_K3698002 is regulatory region of degP, it contains three CpxR sites, which can bound Phosphorylated DNA-binding transcriptional dual regulator CpxR activates transcription of degP. The BBa_K3698002 is degP gene which expresses periplasmic serine endoprotease. This protease is responsible for digesting abnormally folded proteins in E. coli in a high temperature environment to protect E. coli. There is also experimental evidence that DegP is beneficial to E. coli to resist acid stress. For the convenience of characterization, we connected the degP gene fused with chromoprotein after the rDegP. This competent part was inserted into a plasmid containing the chloramphenicol resistance gene for characterization, as shown in Figure 2. Figure1. Schematic diagram of degP_CP circuit Figure2. Plasmid map of pRdegPLCP

Experiment and Results

We first constructed a composite fragment of rDegP and degP. The result is shown in Figure 3. The fragment size is 1757bp. Then the pre-constructed pamilCP was linearized by PCR. The result is shown in Figure 4, and the fragment size is 2876bp. The fragments recovered and purified in the first two steps are transformed into DH5α competent after homologous recombination, and spread on a chloramphenicol-resistant plate, and a single colony is picked for culture. The obtained culture fluid was used for PCR verification, and the primers used were Test-Top and Test-Bottom. After the PCR was completed, gel electrophoresis was performed. The result is shown in Figure 5. The target fragment should be 1000 bp, and the fragment obtained is in line with the expected size, confirming the successful recombination of our pRdegPLCP plasmid. Figure3. Gel electrophoresis of rDegP and degP composite fragments Figure4. Gel electrophoresis of linearized pamilCP fragment Figure5. Gel electrophoresis of pRdegPLCP recombination verifiesThe pRdegPLCP was transformed into DH5α, and placed in normal temperature (37°C) and high temperature (45°C) respectively. The OD588 and OD600 of the bacterial solution were measured every 1h. After 11h, the DH5α_pRdegPLCP at the two temperatures was obtained. The growth curve, as shown in Figure 6, the curve in the high temperature environment is significantly lower than the normal temperature. After enriching the bacterial liquid, it can be seen that the bacterial liquid has a very obvious blue color, as shown in Figure 7. Divide OD588 by OD600 to obtain the absorbance of chromprotein amilCP to characterize the expression of degP, as shown in Figure 7. The expression level of degP at 45°C is obviously higher than that at 37°C. This shows that CpxR in our new part can normally regulate the expression of degP, and can respond to environmental changes. Figure6. Growth curve of DH5α_pRdegPLCP Figure7. Enriched bacteria liquid Figure8. Expression level of degP-amilCP