Difference between revisions of "Part:BBa K5398613"
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Our team has optimized the codon usage of the natural photosensitive DNA-binding protein EL222, originally from the marine bacterium Erythrobacter litoralis HTCC2594, for efficient expression in Escherichia coli. In the dark, EL222 remains in its inactive form, where the N-terminal LOV domain suppresses the DNA-binding activity of its C-terminal HTH domain. Upon exposure to blue light (450nm), the interaction between the LOV and HTH domains is released, allowing the protein to dimerize and bind to its specific DNA target regions. By optimizing the codon usage, we have significantly enhanced its expression in E. coli, providing a more efficient tool for light-controlled gene regulation studies. | Our team has optimized the codon usage of the natural photosensitive DNA-binding protein EL222, originally from the marine bacterium Erythrobacter litoralis HTCC2594, for efficient expression in Escherichia coli. In the dark, EL222 remains in its inactive form, where the N-terminal LOV domain suppresses the DNA-binding activity of its C-terminal HTH domain. Upon exposure to blue light (450nm), the interaction between the LOV and HTH domains is released, allowing the protein to dimerize and bind to its specific DNA target regions. By optimizing the codon usage, we have significantly enhanced its expression in E. coli, providing a more efficient tool for light-controlled gene regulation studies. | ||
| − | < | + | <p>EL222 is a natural photosensitive DNA-binding protein that dimerizes and binds DNA upon blue light exposure. It is composed of a N-terminal light-oxygen-voltage (LOV) domain and a helix-turn-helix DNA-binding domain at the C-terminus. EL222 could act as a transcriptional activator in a tunable blue light-inducible promoter system. pBlind, a synthetic blue light-inducible promoter, is formed by replacing the <i>lux</i> box (LuxR and 3-oxo-C6-HSL complex binding region) which is a 20-bp inverted repeat from the <i>luxI</i> promoter with the 18-bp EL222 binding protein region. When exposed to blue light, EL222 dimerizes and overlaps the -35 region of the promoter thus recruiting RNAP (RNA polymerase). Because the LOV domain is in equilibrium between the dimer and the monomer in the dark state, a high leakage level of this blue light-induced system is witnessed. Aiming to improve the issue, XMU-China 2021 has replaced the strong promoter BBa_J23106 with pBlind. While progress was made, some reporter genes were still expressed without blue light. </p> |
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| + | <html lang="zh"> | ||
| + | <head> | ||
| + | <meta charset="UTF-8"> | ||
| + | <meta name="viewport" content="width=device-width, initial-scale=1.0"> | ||
| + | <style> | ||
| + | .module { | ||
| + | border: 1px solid #ccc; /* 边框 */ | ||
| + | padding: 20px; /* 内边距 */ | ||
| + | margin: 20px auto; /* 外边距，自动居中 */ | ||
| + | width: 600px; /* 模块宽度 */ | ||
| + | text-align: center; /* 内容居中 */ | ||
| + | box-shadow: 0px 0px 10px rgba(0, 0, 0, 0.1); /* 阴影效果 */ | ||
| + | } | ||
| + | </style> | ||
| + | </head> | ||
| + | <body> | ||
| + | <div class="module"> | ||
| + | <img src="https://static.igem.wiki/teams/5398/improvement/output.png" width="500" height="auto" alt="Original EL222 system"> | ||
| + | <p><b> Fig. 1 | EL222 system designed by XMU-China 2021. </b></p> | ||
| + | </div> | ||
| + | </body> | ||
| + | </html> | ||
| + | |||
===Usage and Biology=== | ===Usage and Biology=== | ||
| + | <p>The EL222-based blue light-inducible system allows precise control of gene expression by dimerizing and binding DNA upon blue light exposure. To reduce system leakage, a LicV fusion protein and RAT sequence were introduced, enhancing control by preventing premature transcription termination. This system is ideal for applications requiring tight regulation of gene expression in response to light, such as biosensors and metabolic engineering.</p> | ||
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Latest revision as of 13:21, 2 October 2024
Codon-Optimized EL222 Regulatory Element, improved from Part:BBa_K2332004
Our team has optimized the codon usage of the natural photosensitive DNA-binding protein EL222, originally from the marine bacterium Erythrobacter litoralis HTCC2594, for efficient expression in Escherichia coli. In the dark, EL222 remains in its inactive form, where the N-terminal LOV domain suppresses the DNA-binding activity of its C-terminal HTH domain. Upon exposure to blue light (450nm), the interaction between the LOV and HTH domains is released, allowing the protein to dimerize and bind to its specific DNA target regions. By optimizing the codon usage, we have significantly enhanced its expression in E. coli, providing a more efficient tool for light-controlled gene regulation studies.
EL222 is a natural photosensitive DNA-binding protein that dimerizes and binds DNA upon blue light exposure. It is composed of a N-terminal light-oxygen-voltage (LOV) domain and a helix-turn-helix DNA-binding domain at the C-terminus. EL222 could act as a transcriptional activator in a tunable blue light-inducible promoter system. pBlind, a synthetic blue light-inducible promoter, is formed by replacing the lux box (LuxR and 3-oxo-C6-HSL complex binding region) which is a 20-bp inverted repeat from the luxI promoter with the 18-bp EL222 binding protein region. When exposed to blue light, EL222 dimerizes and overlaps the -35 region of the promoter thus recruiting RNAP (RNA polymerase). Because the LOV domain is in equilibrium between the dimer and the monomer in the dark state, a high leakage level of this blue light-induced system is witnessed. Aiming to improve the issue, XMU-China 2021 has replaced the strong promoter BBa_J23106 with pBlind. While progress was made, some reporter genes were still expressed without blue light.
Fig. 1 | EL222 system designed by XMU-China 2021.
Usage and Biology
The EL222-based blue light-inducible system allows precise control of gene expression by dimerizing and binding DNA upon blue light exposure. To reduce system leakage, a LicV fusion protein and RAT sequence were introduced, enhancing control by preventing premature transcription termination. This system is ideal for applications requiring tight regulation of gene expression in response to light, such as biosensors and metabolic engineering.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 320
Illegal BamHI site found at 138 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 196
Illegal AgeI site found at 306 - 1000COMPATIBLE WITH RFC[1000]