Difference between revisions of "Part:BBa K2374003:Experience"
(→Design notes) |
|||
| (3 intermediate revisions by the same user not shown) | |||
| Line 5: | Line 5: | ||
===Applications of BBa_K2374003=== | ===Applications of BBa_K2374003=== | ||
| + | ===Design notes=== | ||
| + | [[File:2017tongji_image_registry_ple4.png|right|200px|pleP-GAL4]] | ||
| + | [[File:2017tongji_image_registry_ple80.png|right|200px|pleP-GAL80ts]] | ||
| + | [[File:2017tongji_image_registry_uTH.png|right|200px|pleP-GAL80ts]] | ||
| + | We cloned synthetic TH into pUAST with restriction endonuclease digestion and T4 ligase igation. Then we construct pSB1C3-UAS-TH and pUAST-UAS-TH. The pSB1C3-UAS-TH is for submission. The pUAST-UAS-TH also with the other two plasmids: pUAST-ple-GAL4 ([https://parts.igem.org/Part:BBa_K2374005 BBa_K2374005])and pUAST-ple-GAL80ts ([https://parts.igem.org/Part:BBa_K2374006 BBa_K2374006]) are used to do micro-injection into the ''D.melanogaster''. We must combine the three pathways to determine if the system work well. After hybridization screening, we got stable modified fruit fly strains. Finally, we did RT-PCR, qPCR and behavioral experiments to test our system. <br>The result of our testing on ''D.melanogaster'' is displayed below. | ||
| + | '''''note''''': | ||
| + | 1. At 18-25°C (the optimum temperature for fruit flies’ growth), it has the activity binding to Gal4, which will eliminate the effect Gal4 binding to UAS, then downstream gene TH will not express and the expression level of dopamine is normal.<br> | ||
| + | 2. When the temperature is up to 29℃, Gal80ts will be inactivated, then Gal4 works properly, binding to Gal4 binding sequence on UAS, and start the expression of downstream gene TH which will leads to the overexpression of dopamine in ''Drosophila''.<br> | ||
| + | [http://2017.igem.org/Team:Tongji_China/Design More Information] | ||
| + | |||
| + | ==Test Results== | ||
| + | 1. Use Real-time PCR to detect whether the expression of TH is increased at 29°C. It shows that the relative expression of TH in modified fruit flies increased significantly.<br> | ||
| + | [[File:2017tongji_image_registry_qPCR.png|center|300px]] | ||
| + | 2. Detect male-male courtship when raising the temperature. Mating index refers to the relative time that the fruit fly use for mating.<br> | ||
| + | It shows that he mating index of the treated group rises significantly in at 29°C. [time=5minutes, n=5, P<0.01] | ||
| + | [[File:2017tongji_image_registry_behavior1.png|center|350px]] | ||
| + | |||
| + | [http://2017.igem.org/Team:Tongji_China/Experiments More details]'''About Graphs and Videos''' | ||
===User Reviews=== | ===User Reviews=== | ||
<!-- DON'T DELETE --><partinfo>BBa_K2374003 StartReviews</partinfo> | <!-- DON'T DELETE --><partinfo>BBa_K2374003 StartReviews</partinfo> | ||
Latest revision as of 20:00, 30 October 2017
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_K2374003
Design notes
We cloned synthetic TH into pUAST with restriction endonuclease digestion and T4 ligase igation. Then we construct pSB1C3-UAS-TH and pUAST-UAS-TH. The pSB1C3-UAS-TH is for submission. The pUAST-UAS-TH also with the other two plasmids: pUAST-ple-GAL4 (BBa_K2374005)and pUAST-ple-GAL80ts (BBa_K2374006) are used to do micro-injection into the D.melanogaster. We must combine the three pathways to determine if the system work well. After hybridization screening, we got stable modified fruit fly strains. Finally, we did RT-PCR, qPCR and behavioral experiments to test our system.
The result of our testing on D.melanogaster is displayed below.
note:
1. At 18-25°C (the optimum temperature for fruit flies’ growth), it has the activity binding to Gal4, which will eliminate the effect Gal4 binding to UAS, then downstream gene TH will not express and the expression level of dopamine is normal.
2. When the temperature is up to 29℃, Gal80ts will be inactivated, then Gal4 works properly, binding to Gal4 binding sequence on UAS, and start the expression of downstream gene TH which will leads to the overexpression of dopamine in Drosophila.
[http://2017.igem.org/Team:Tongji_China/Design More Information]
Test Results
1. Use Real-time PCR to detect whether the expression of TH is increased at 29°C. It shows that the relative expression of TH in modified fruit flies increased significantly.
2. Detect male-male courtship when raising the temperature. Mating index refers to the relative time that the fruit fly use for mating.
It shows that he mating index of the treated group rises significantly in at 29°C. [time=5minutes, n=5, P<0.01]
[http://2017.igem.org/Team:Tongji_China/Experiments More details]About Graphs and Videos
User Reviews
UNIQ2a4b8883d13c148d-partinfo-00000000-QINU UNIQ2a4b8883d13c148d-partinfo-00000001-QINU