Difference between revisions of "Part:BBa K3502011"
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| − | This year, we tested the editing efficiency of ADAR1 by adding a pTRE system and a stem ring. The essence grains were added with fluorescent protein after the pTRE promoter to report the editing efficiency. In this system, it is hoped that ADAR1 will edit the editing sites on the stem ring to suppress downstream toxic proteins expression | + | This year, we tested the editing efficiency of ADAR1 by adding a pTRE system and a stem ring. The essence grains were added with fluorescent protein after the pTRE promoter to report the editing efficiency.The upstream of the stem ring is GFP, and the downstream is toxic protein In this system, it is hoped that ADAR1 will edit the editing sites on the stem ring to suppress downstream toxic proteins expression. |
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| − | In order to know how many DOX our system needs to work effectively, and how the stem-loop function to this system. we do an experiment about the relation between the concentration of DOX and GFP expression, which shows the induced expression efficiency of pTRE was demonstrated | + | In order to know how many DOX our system needs to work effectively, and how the stem-loop function to this system. we do an experiment about the relation between the concentration of DOX and GFP expression, which shows the induced expression efficiency of pTRE was demonstrated. We calculated the effect of this system by looking at fluorescence and cell count |
[[File:T--SYSU-CHINA--contri-p1.png|500px|thumb|left]] | [[File:T--SYSU-CHINA--contri-p1.png|500px|thumb|left]] | ||
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Revision as of 03:29, 28 October 2020
a GFP-STEM-LOOP-apoptin vector controlled by TRE-on system
This year, we tested the editing efficiency of ADAR1 by adding a pTRE system and a stem ring. The essence grains were added with fluorescent protein after the pTRE promoter to report the editing efficiency.The upstream of the stem ring is GFP, and the downstream is toxic protein In this system, it is hoped that ADAR1 will edit the editing sites on the stem ring to suppress downstream toxic proteins expression.
In order to know how many DOX our system needs to work effectively, and how the stem-loop function to this system. we do an experiment about the relation between the concentration of DOX and GFP expression, which shows the induced expression efficiency of pTRE was demonstrated. We calculated the effect of this system by looking at fluorescence and cell count
We transferred the plasmid contains STEM-Loop into HeLa cells (with high endogenous ADAR1 expression), and noticed that plasmid with stem ring was effectively inhibited when Dox was low, indicating the success of ADAR1 in stem ring editing.Since the inhibitory effect of ADAR1 was relieved at high Dox expression, we established the specification for experiments at low DOX-induced concentration
We also noted that the introduction of the stem-loop effectively reduced the leakage expression of pTRE at low Dox concentration and had little effect on the expression at high concentration, if the efficiency assessment of the Tet-on system was applied alone.Since ADAR1 is widely present in various commonly used cell lines (such as HeLa, Hep2, etc.), we believe that the pTRE promoter binding to stem rings can be an important improvement of the pTRE system.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1348
Illegal BamHI site found at 10 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1015