Difference between revisions of "Part:BBa K2332004"
 
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__NOTOC__
 
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<partinfo>BBa_K2332004 short</partinfo>
 
<partinfo>BBa_K2332004 short</partinfo>
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EL222 is a natural photosensitive DNA-binding protein from the marine bacterium Erythrobacter litoralis HTCC2594. In the dark, EL222 is in its inactive form where its N-terminal LOV domain represses its DNA-binding C-terminal HTH domain. Upon blue light exposure (450nm), LOV-HTH interaction is released, allowing it to dimerize and bind its DNA binding regions.
 
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===Usage and Biology===
 
===Usage and Biology===
 +
 +
EL222 is a natural photosensitive DNA-binding protein from the marine bacterium Erythrobacter litoralis HTCC2594. In the dark, EL222 is in its inactive form where its N-terminal LOV domain represses its DNA-binding C-terminal HTH domain. Upon blue light exposure (450nm), LOV-HTH interaction is released, allowing it to dimerize and bind its DNA binding regions.
  
 
==Characterization from XMU-China 2021==
 
==Characterization from XMU-China 2021==
 +
'''Group''': [https://2020.igem.org/Team/XMU-China iGEM Team XMU-China 2020]
  
 +
===Characterization===
 
In some designs of pBLind-EL222 system, EL222 was usually expressed by a constitutive promoter in the Anderson family, such as <partinfo>BBa_J23119</partinfo> and <partinfo>BBa_J23106</partinfo>. Although the EL222 protein is activated to bind pBLind promoter due to the conformational change upon blue light illumination, the binding events still happen by chance in dark environment, which results in unexpected transcriptional leakage of the genes controlled by pBLind promoter. Reducing the existent pool of EL222 protein in the cell will low down the leakage level in the dark state. Therefore, using an inducible promoter to control the expression of EL222 seems to be an ideal option to lower the leakage when the blue-light irradiation is not supplied.
 
In some designs of pBLind-EL222 system, EL222 was usually expressed by a constitutive promoter in the Anderson family, such as <partinfo>BBa_J23119</partinfo> and <partinfo>BBa_J23106</partinfo>. Although the EL222 protein is activated to bind pBLind promoter due to the conformational change upon blue light illumination, the binding events still happen by chance in dark environment, which results in unexpected transcriptional leakage of the genes controlled by pBLind promoter. Reducing the existent pool of EL222 protein in the cell will low down the leakage level in the dark state. Therefore, using an inducible promoter to control the expression of EL222 seems to be an ideal option to lower the leakage when the blue-light irradiation is not supplied.
  
 
[[File:T--XMU-China--EL222_contribution.jpg|800px|center]]
 
[[File:T--XMU-China--EL222_contribution.jpg|800px|center]]
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Figure 3: The illustration and characterization of the system. (A)Gene circuit illustration for the system.(B) RFUsfGFP/OD600 of the system in dark and blue-light condition was calculated as time progressed.
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'''Fig. 1.''' The illustration and characterization of the system. (A)Gene circuit illustration for the system.(B) RFUsfGFP/OD<sub>600</sub> of the system in dark and blue-light condition was calculated as time progressed.
 +
 
 +
The result showed that the system maintained a well blue-light sensitivity and a higher expression level of sfGFP in the system was observed as time progressed (Fig. 1B). It demonstrated that the EL222 was able to work as a photosensitive DNA-binding protein and pBLind could be activated by EL222.
 +
 
 +
== Characterisation by HKUST-GZ 2024 ==
 +
'''Group''': [https://2024.igem.wiki/hkust-gz/ HKUST-GZ]
 +
 
 +
'''Author''': Hua XU
 +
 
 +
=== Cell Toxicity ===
 +
 
 +
Some scientists have report that EL222 may cause cell toxicity (see the paper DOI: 10.34133/2021/9857418). This year, our HKUST-GZ iGEM team investigated into this issues. We transformed the light sensor into E. coli cells, and found that the growth curve is basically identical to cells transformed with empty vectors. Further experiments neeed to be done to fully understand the mechanism behind the cell toxicity phenomenon.
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<html>
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<head>
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    <style>
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        body {
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            font-family: Arial, sans-serif; /* Set a clean font */
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            background-color: #eaeaea; /* Light background for the entire page */
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            color: #333; /* Dark text color for better readability */
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            padding: 20px; /* Space around the content */
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        }
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html
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    .figure-box {
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        border: 2px solid #007BFF; /* Blue border for a modern look */
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        padding: 15px; /* Space inside the box */
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        margin: 20px; /* Space outside the box */
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        display: inline-block; /* Keep the box only as wide as its content */
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        border-radius: 10px; /* More rounded corners */
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        box-shadow: 0 4px 20px rgba(0, 0, 0, 0.2); /* Deeper shadow for depth */
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        background-color: #ffffff; /* White background for the box */
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        transition: box-shadow 0.3s ease; /* Smooth transition for hover effect */
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    }
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    .figure-box:hover {
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        box-shadow: 0 8px 40px rgba(0, 0, 0, 0.3); /* Darker shadow on hover */
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    }
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    .figure-box img {
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        width: 100%; /* Make image responsive */
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        height: auto; /* Maintain aspect ratio */
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        border-radius: 5px; /* Slightly round the image corners */
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    }
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    .figure-box p {
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        margin-top: 15px; /* Space between image and text */
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        line-height: 1.6; /* Improved line spacing for readability */
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    }
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</style>
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</head>
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</html>
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<html>
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<body>
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    <div class="figure-box">
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        <img
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            src="https://static.igem.wiki/teams/5280/registry/lexro/cell-toxicity-with-el-final-version.png"
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            style="width:800px; display:block; margin:0;"
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            alt="In the experiments, E. coli transformed with an empty vector were used as negative controls."
 +
        >
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        <p>
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            Cultivated cells transformed with different plasmids under optimal conditions were synchronized, and samples were taken every 10 minutes to measure the OD600 of the cells.In this figure, LexRO (<partinfo>BBa_K5280129</partinfo>) is another light sensor similar to the functionalities of EL222.
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        </p>
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    </div>
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</body>
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</html>
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 +
=== Fluorescence ===
 +
 
 +
It has been reported that EL222 has emssion light when exposed to light with 450nm wavelength.
 +
For more information, you can refer to the paper DOI: 10.34133/2021/9857418.
  
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The result showed that the system maintained a well blue-light sensitivity and a higher expression level of sfGFP in the system was observed as time progressed (Fig. 3B). It demonstrated that the EL222 was able to work as a photosensitive DNA-binding protein and pBLind could be activated by EL222.
 
  
 
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Latest revision as of 14:10, 29 September 2024


EL222, photosensitive DNA-binding protein

Usage and Biology

EL222 is a natural photosensitive DNA-binding protein from the marine bacterium Erythrobacter litoralis HTCC2594. In the dark, EL222 is in its inactive form where its N-terminal LOV domain represses its DNA-binding C-terminal HTH domain. Upon blue light exposure (450nm), LOV-HTH interaction is released, allowing it to dimerize and bind its DNA binding regions.

Characterization from XMU-China 2021

Group: iGEM Team XMU-China 2020

Characterization

In some designs of pBLind-EL222 system, EL222 was usually expressed by a constitutive promoter in the Anderson family, such as BBa_J23119 and BBa_J23106. Although the EL222 protein is activated to bind pBLind promoter due to the conformational change upon blue light illumination, the binding events still happen by chance in dark environment, which results in unexpected transcriptional leakage of the genes controlled by pBLind promoter. Reducing the existent pool of EL222 protein in the cell will low down the leakage level in the dark state. Therefore, using an inducible promoter to control the expression of EL222 seems to be an ideal option to lower the leakage when the blue-light irradiation is not supplied.

T--XMU-China--EL222 contribution.jpg

Fig. 1. The illustration and characterization of the system. (A)Gene circuit illustration for the system.(B) RFUsfGFP/OD600 of the system in dark and blue-light condition was calculated as time progressed.

The result showed that the system maintained a well blue-light sensitivity and a higher expression level of sfGFP in the system was observed as time progressed (Fig. 1B). It demonstrated that the EL222 was able to work as a photosensitive DNA-binding protein and pBLind could be activated by EL222.

Characterisation by HKUST-GZ 2024

Group: HKUST-GZ

Author: Hua XU

Cell Toxicity

Some scientists have report that EL222 may cause cell toxicity (see the paper DOI: 10.34133/2021/9857418). This year, our HKUST-GZ iGEM team investigated into this issues. We transformed the light sensor into E. coli cells, and found that the growth curve is basically identical to cells transformed with empty vectors. Further experiments neeed to be done to fully understand the mechanism behind the cell toxicity phenomenon.

In the experiments, E. coli transformed with an empty vector were used as negative controls.

Cultivated cells transformed with different plasmids under optimal conditions were synchronized, and samples were taken every 10 minutes to measure the OD600 of the cells.In this figure, LexRO (BBa_K5280129) is another light sensor similar to the functionalities of EL222.

Fluorescence

It has been reported that EL222 has emssion light when exposed to light with 450nm wavelength. For more information, you can refer to the paper DOI: 10.34133/2021/9857418.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 81
    Illegal AgeI site found at 306
  • 1000
    COMPATIBLE WITH RFC[1000]