Difference between revisions of "Part:BBa K5490024"
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| − | + | It’s a luciferase reporter gene with three 23-base-long sequences that can be targeted by endonuclease enzymes such as RNAi or Cas13. The reporter can be analyzed either through microscopic observation, as it is fused with an unstable EGFP, or through a luciferase assay to evaluate the interaction between the endonuclease and the target sites. | |
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<partinfo>BBa_K5490024 parameters</partinfo> | <partinfo>BBa_K5490024 parameters</partinfo> | ||
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| + | This composite part consists of two distinct reporter systems designed for effective monitoring of gene activity. Upstream is an unstable eGFP reporter, which is ideal for live-cell imaging, allowing researchers to visualize cellular processes in real time. Downstream, there is a luciferase gene fused with degradation sites to enhance the responsiveness of the signal. | ||
| + | |||
| + | The luciferase component features three target sites, each 23 nucleotides in length, that are found within the WNV genome. Two of these target sites are located upstream of a flexible joint, while the third is situated just upstream of the poly(A) signal. These sequences have been selected using an algorithmic approach, identifying them as optimal targets for the effector molecule CasRx. | ||
| + | |||
| + | One of the advantages of this composite design is the flexibility it offers researchers: they can easily swap out these target sequences for any sequences they wish to target using RNAi or Cas13. The unstable nature of both reporters ensures that the reporter signal will cease almost immediately after cleavage occurs, providing a rapid and clear indication of gene activity. | ||
| + | |||
| + | By employing two reporters, this system enables dual-method validation. Researchers can assess gene expression in real time using live-cell imaging under a microscope, while also conducting quantitative measurements in a luciferase assay post-cleavage. This combination of methodologies enhances the robustness of experimental results, allowing for comprehensive analysis of gene regulation and interference efficiency. | ||
| + | |||
| + | Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, Molin S. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol. 1998 Jun;64(6):2240-6. doi: 10.1128/AEM.64.6.2240-2246.1998. PMID: 9603842; PMCID: PMC106306. | ||
| + | Huttly A. Reporter genes. Methods Mol Biol. 2009;478:39-69. doi: 10.1007/978-1-59745-379-0_3. PMID: 19009438. | ||
| + | Meyer RD, Srinivasan S, Singh AJ, Mahoney JE, Gharahassanlou KR, Rahimi N. PEST motif serine and tyrosine phosphorylation controls vascular endothelial growth factor receptor 2 stability and downregulation. Mol Cell Biol. 2011 May;31(10):2010-25. doi: 10.1128/MCB.01006-10. Epub 2011 Mar 14. PMID: 21402774; PMCID: PMC3133358. | ||
| + | Conti E, Franks NP, Brick P. Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure. 1996 Mar 15;4(3):287-98. doi: 10.1016/s0969-2126(96)00033-0. PMID: 8805533. | ||
| + | Chng J, Wang T, Nian R, Lau A, Hoi KM, Ho SC, Gagnon P, Bi X, Yang Y. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells. MAbs. 2015;7(2):403-12. doi: 10.1080/19420862.2015.1008351. PMID: 25621616; PMCID: PMC4622431. | ||
Revision as of 23:54, 28 September 2024
REPORTER FOR TESTING WNV GRNA TARGET SITES
It’s a luciferase reporter gene with three 23-base-long sequences that can be targeted by endonuclease enzymes such as RNAi or Cas13. The reporter can be analyzed either through microscopic observation, as it is fused with an unstable EGFP, or through a luciferase assay to evaluate the interaction between the endonuclease and the target sites.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 3271
Illegal XbaI site found at 3493
Illegal PstI site found at 1490 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 3271
Illegal PstI site found at 1490
Illegal NotI site found at 3476 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 3271
Illegal BamHI site found at 3484 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 3271
Illegal XbaI site found at 3493
Illegal PstI site found at 1490 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 3271
Illegal XbaI site found at 3493
Illegal PstI site found at 1490
Illegal NgoMIV site found at 1652
Illegal NgoMIV site found at 3029
Illegal NgoMIV site found at 3050
Illegal NgoMIV site found at 3365
Illegal AgeI site found at 2744 - 1000COMPATIBLE WITH RFC[1000]
This composite part consists of two distinct reporter systems designed for effective monitoring of gene activity. Upstream is an unstable eGFP reporter, which is ideal for live-cell imaging, allowing researchers to visualize cellular processes in real time. Downstream, there is a luciferase gene fused with degradation sites to enhance the responsiveness of the signal.
The luciferase component features three target sites, each 23 nucleotides in length, that are found within the WNV genome. Two of these target sites are located upstream of a flexible joint, while the third is situated just upstream of the poly(A) signal. These sequences have been selected using an algorithmic approach, identifying them as optimal targets for the effector molecule CasRx.
One of the advantages of this composite design is the flexibility it offers researchers: they can easily swap out these target sequences for any sequences they wish to target using RNAi or Cas13. The unstable nature of both reporters ensures that the reporter signal will cease almost immediately after cleavage occurs, providing a rapid and clear indication of gene activity.
By employing two reporters, this system enables dual-method validation. Researchers can assess gene expression in real time using live-cell imaging under a microscope, while also conducting quantitative measurements in a luciferase assay post-cleavage. This combination of methodologies enhances the robustness of experimental results, allowing for comprehensive analysis of gene regulation and interference efficiency.
Andersen JB, Sternberg C, Poulsen LK, Bjorn SP, Givskov M, Molin S. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl Environ Microbiol. 1998 Jun;64(6):2240-6. doi: 10.1128/AEM.64.6.2240-2246.1998. PMID: 9603842; PMCID: PMC106306. Huttly A. Reporter genes. Methods Mol Biol. 2009;478:39-69. doi: 10.1007/978-1-59745-379-0_3. PMID: 19009438. Meyer RD, Srinivasan S, Singh AJ, Mahoney JE, Gharahassanlou KR, Rahimi N. PEST motif serine and tyrosine phosphorylation controls vascular endothelial growth factor receptor 2 stability and downregulation. Mol Cell Biol. 2011 May;31(10):2010-25. doi: 10.1128/MCB.01006-10. Epub 2011 Mar 14. PMID: 21402774; PMCID: PMC3133358. Conti E, Franks NP, Brick P. Crystal structure of firefly luciferase throws light on a superfamily of adenylate-forming enzymes. Structure. 1996 Mar 15;4(3):287-98. doi: 10.1016/s0969-2126(96)00033-0. PMID: 8805533. Chng J, Wang T, Nian R, Lau A, Hoi KM, Ho SC, Gagnon P, Bi X, Yang Y. Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells. MAbs. 2015;7(2):403-12. doi: 10.1080/19420862.2015.1008351. PMID: 25621616; PMCID: PMC4622431.