Difference between revisions of "Part:BBa K511401"
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This part encodes a fusion protein consisting of the human HRH4 receptor, the recognition sequence for the tobacco etch virus (TEV) protease, and the Gal4-VP16 transactivator. | This part encodes a fusion protein consisting of the human HRH4 receptor, the recognition sequence for the tobacco etch virus (TEV) protease, and the Gal4-VP16 transactivator. | ||
| − | < | + | ===Improvement=== |
| − | + | <p>In our project, human histamine receptor H4 (HRH4) with mcherry tag under the control of a constitutive promoter—ADH1 promoter in yeast. The function is testing the expression strength and location of human histamine receptor H4 (HRH4) in yeast. | |
| + | 1.Fluorescence microscopy | ||
| + | After transforming the CENPK2-1C ura3::far1 his2::sst2 trp3::ste2 △ura3 Saccharomyces cerevisiae with plasmid pGADT7 contains part BBa_K2583001, 30℃ cultivate on △leu solid medium for one day. Select monoclonal and enlarge culture overnight, then observe the fluorescence under the microscope with 580nm exciting light. The red fluorescence shows the expression of HRH4-mcherry fusion protein. | ||
| + | 2.Flow CytoMetry (FCM) | ||
| + | From the result of fluorescence microscopy, the expression of HRH4-mCherry fusion proteins can be observed roughly, for quantifying the expression level of HRH4-mCherry, FCM is performed. From the result, a distinct peak value can be observed. Considering the fluorescence microscopy result that most of HRH4 are correctly located, this data does provide us a reliable probability distribution of total H4 protein expression variance between yeast individuals. Finally, the probability distribution of functional H4 receptor expression variance can refer to the total expression probability distribution we get from experiments | ||
| + | [[Image:T--Tsinghua-A--mcherry_tagged_HRH4_1.png|960px]] | ||
| + | Fig1. Fluorescence microscopic result of HRH4-mcherry | ||
| + | [[Image:T--Tsinghua-A--mcherry_tagged_HRH4_2.png|960px]] | ||
| + | Fig2. Fluorescence microscopic result of DIO dying and HRH4-mcherry | ||
| + | [[Image:T--Tsinghua-A--mcherry_tagged_HRH4_3.png|960px]] | ||
| + | Fig3. Flow Cytometry result of HRH4-mCherry fusion protein expression | ||
| + | </p> | ||
| + | |||
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Latest revision as of 03:18, 18 October 2018
HRH4-TEVs-GV16 Signaling Receptor MammoBlock
This part encodes a fusion protein consisting of the human HRH4 receptor, the recognition sequence for the tobacco etch virus (TEV) protease, and the Gal4-VP16 transactivator.
Improvement
In our project, human histamine receptor H4 (HRH4) with mcherry tag under the control of a constitutive promoter—ADH1 promoter in yeast. The function is testing the expression strength and location of human histamine receptor H4 (HRH4) in yeast.
1.Fluorescence microscopy
After transforming the CENPK2-1C ura3::far1 his2::sst2 trp3::ste2 △ura3 Saccharomyces cerevisiae with plasmid pGADT7 contains part BBa_K2583001, 30℃ cultivate on △leu solid medium for one day. Select monoclonal and enlarge culture overnight, then observe the fluorescence under the microscope with 580nm exciting light. The red fluorescence shows the expression of HRH4-mcherry fusion protein.
2.Flow CytoMetry (FCM)
From the result of fluorescence microscopy, the expression of HRH4-mCherry fusion proteins can be observed roughly, for quantifying the expression level of HRH4-mCherry, FCM is performed. From the result, a distinct peak value can be observed. Considering the fluorescence microscopy result that most of HRH4 are correctly located, this data does provide us a reliable probability distribution of total H4 protein expression variance between yeast individuals. Finally, the probability distribution of functional H4 receptor expression variance can refer to the total expression probability distribution we get from experiments
Fig1. Fluorescence microscopic result of HRH4-mcherry
Fig2. Fluorescence microscopic result of DIO dying and HRH4-mcherry
Fig3. Flow Cytometry result of HRH4-mCherry fusion protein expression
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 551
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 551
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 551
Illegal BglII site found at 713
Illegal BamHI site found at 1178
Illegal BamHI site found at 1208
Illegal XhoI site found at 1674 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 551
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 551
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1593
Illegal SapI site found at 863