Difference between revisions of "Part:BBa K1993019"
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T2A is a self-cleaving 2A peptide (The average length is 18–22 amino acids) that could be applied in expression of more than one gene in cells. It is a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. [1] | T2A is a self-cleaving 2A peptide (The average length is 18–22 amino acids) that could be applied in expression of more than one gene in cells. It is a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. [1] | ||
| − | In our project, we found that protein coding genes downstream were not expressed if they followed another protein coding gene directly. Taking advantage of function of T2A, we constructed a plasmid that T2A linked between two protein coding genes. For example, we constructed BBa_K1993006 (Luciferase-T2A-dTomato-T2A-hFTH) to ensure luciferase, dTomato and hFTH could be expressed. | + | In our project, we found that protein coding genes downstream were not expressed if they followed another protein coding gene directly. Taking advantage of function of T2A, we constructed a plasmid that T2A linked between two protein coding genes. For example, we constructed [https://parts.igem.org/Part:BBa_K1993006 BBa_K1993006](Luciferase-T2A-dTomato-T2A-hFTH) to ensure luciferase, dTomato and hFTH could be expressed. |
==References== | ==References== | ||
[1] Kim J H, Lee S R, Li L H, et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. [J]. Plos One, 2011, 6(4): e18556. | [1] Kim J H, Lee S R, Li L H, et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. [J]. Plos One, 2011, 6(4): e18556. | ||
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| + | ===improvement by CPU_CHINA 2019=== | ||
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| + | [[File:T--CPU_CHINA--part improvement.png|850px|thumb|center|Figure 1. Flow cytometry analysis of CD14 expression in cells without transfection, transfected with plasmid (TLR1-T2A-CD14) and transfected with plasmid (TLR1-improved T2A-CD14).]] | ||
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| + | <p>In order to confirm the cleavage efficiency of the GSG-added T2A was improved, T2A (BBa_K1993019) and improved T2A ([[Part:BBa_K2976010]]) were separately constructed into expression vector for co-expression of TLR1 and CD14. And we performed flow cytometry on cells transfected with plasmid (TLR1-T2A-CD14) and plasmid (TLR1-improved T2A-CD14). According to Figure 1, the amount of CD14 in the cells that were transfected with plasmid (TLR1-improved T2A-CD14) was higher than that of the plasmid (TLR1-T2A-CD14), which proved that the cleavage efficiency of the part is enhanced, and verified our improved part achieved the expected functionality.</p> | ||
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<!-- Add more about the biology of this part here | <!-- Add more about the biology of this part here | ||
Latest revision as of 08:04, 23 July 2021
T2A
T2A is a self-cleaving 2A peptide (The average length is 18–22 amino acids) that could be applied in expression of more than one gene in cells. It is a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. [1]
In our project, we found that protein coding genes downstream were not expressed if they followed another protein coding gene directly. Taking advantage of function of T2A, we constructed a plasmid that T2A linked between two protein coding genes. For example, we constructed BBa_K1993006(Luciferase-T2A-dTomato-T2A-hFTH) to ensure luciferase, dTomato and hFTH could be expressed.
References
[1] Kim J H, Lee S R, Li L H, et al. High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. [J]. Plos One, 2011, 6(4): e18556.
improvement by CPU_CHINA 2019
In order to confirm the cleavage efficiency of the GSG-added T2A was improved, T2A (BBa_K1993019) and improved T2A (Part:BBa_K2976010) were separately constructed into expression vector for co-expression of TLR1 and CD14. And we performed flow cytometry on cells transfected with plasmid (TLR1-T2A-CD14) and plasmid (TLR1-improved T2A-CD14). According to Figure 1, the amount of CD14 in the cells that were transfected with plasmid (TLR1-improved T2A-CD14) was higher than that of the plasmid (TLR1-T2A-CD14), which proved that the cleavage efficiency of the part is enhanced, and verified our improved part achieved the expected functionality.
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]