Difference between revisions of "Part:BBa K4140017"
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==Part Description== | ==Part Description== | ||
| − | CMV (cytomegalovirus) promoter is a | + | CMV (cytomegalovirus) promoter is a strong constitutive promoter commonly used in mammalian expression systems to produce high levels of expression, However CMV promoter is one of the most variable types of promoter as it is highly variable between different cell types such as being strong in some cell types and being much weaker in other types |
| − | + | ||
==Usage== | ==Usage== | ||
| − | + | We used a mammalian expression systems to regulate the expression of Cas12g which have a remarkable role on tuning and controlling our circuit activity and the levels of PAH or beta-galactosidase in extreme conditions acting as an autoregulation systems as shown in figure 1. | |
| − | + | [[File:reg.png|thumb|Right|Figure 1. (shows SBOL of the usage of CMV in our circuit.) ]] | |
| + | <br><br><br><br><br><br><br><br> | ||
==Literature Characterization== | ==Literature Characterization== | ||
| + | An essential part originated from the human cytomegalovirus we employ it to regulate the expression of the Cas12g the protein domain of our CRISPR regulatory system CMV promoter is known to have a moderate level of expression lesser than other promoters we take advantage of that to limit the possibility of overexpression of Cas12g to reduce the off targeting effect | ||
| + | [[File:CMV-1.png|thumb|right|Figure.2 Effect of different promoter on transfection efficiency and transient transgene expression using eGFP antibody analysis.]] | ||
| + | <br><br> | ||
| + | The greatest expression levels were seen in the cells transfected with CHEF-1 promoter-carrying vectors, followed by those with HEF-1, CMV mutant, CMV, mouse CMV, CAG, CAG enhancer, and PGK. When the amount of eGFP expression under the CMV promoter was taken into account as 100, the levels of eGFP expression under the PGK, CHEF-1, HEF-1, mouse CMV, CMV mutant, CAG, and CAG enhancer promoters were 218.13, 184.21, 164.33, 49.12, 47.95, and 8.77, respectively. | ||
| + | <br><br><br><br><br><br><br><br><br><br><br><br><br><br><br> | ||
| + | ==Experimental Characterization== | ||
| + | [[File:capture7.png|right|]] | ||
| + | <br><br><br><br><br> | ||
| + | This figure shows an experimental characterization of this part as it's validated through gel electrophoresis as it is in lane 4. The running part (ordered from IDT) included Human u6 Promoter - -Kinkturn - CMV Promoter - Cas12g. | ||
| + | <br><br><br><br><br><br><br><br><br><br><br><br> | ||
| − | + | ==References== | |
| − | + | 1. Pham, P. L., Kamen, A. & Durocher, Y. Large-scale transfection of mammalian cells for the fast production of recombinant protein. Mol. Biotechnol. 34, 225–237 (2006). | |
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===Usage and Biology=== | ===Usage and Biology=== | ||
Latest revision as of 04:27, 12 October 2022
CMV promoter
Part Description
CMV (cytomegalovirus) promoter is a strong constitutive promoter commonly used in mammalian expression systems to produce high levels of expression, However CMV promoter is one of the most variable types of promoter as it is highly variable between different cell types such as being strong in some cell types and being much weaker in other types
Usage
We used a mammalian expression systems to regulate the expression of Cas12g which have a remarkable role on tuning and controlling our circuit activity and the levels of PAH or beta-galactosidase in extreme conditions acting as an autoregulation systems as shown in figure 1.
Literature Characterization
An essential part originated from the human cytomegalovirus we employ it to regulate the expression of the Cas12g the protein domain of our CRISPR regulatory system CMV promoter is known to have a moderate level of expression lesser than other promoters we take advantage of that to limit the possibility of overexpression of Cas12g to reduce the off targeting effect
The greatest expression levels were seen in the cells transfected with CHEF-1 promoter-carrying vectors, followed by those with HEF-1, CMV mutant, CMV, mouse CMV, CAG, CAG enhancer, and PGK. When the amount of eGFP expression under the CMV promoter was taken into account as 100, the levels of eGFP expression under the PGK, CHEF-1, HEF-1, mouse CMV, CMV mutant, CAG, and CAG enhancer promoters were 218.13, 184.21, 164.33, 49.12, 47.95, and 8.77, respectively.
Experimental Characterization
This figure shows an experimental characterization of this part as it's validated through gel electrophoresis as it is in lane 4. The running part (ordered from IDT) included Human u6 Promoter - -Kinkturn - CMV Promoter - Cas12g.
References
1. Pham, P. L., Kamen, A. & Durocher, Y. Large-scale transfection of mammalian cells for the fast production of recombinant protein. Mol. Biotechnol. 34, 225–237 (2006).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]