Difference between revisions of "Part:BBa K2374005"

Revision as of 18:02, 31 October 2017

TH-GAL4

The expression of GAL4 is controlled by TH(ple) promoter.

Design notes

ple has three alternatively spliced transcript variants which encode iosforms of TH. We choose isoform B to construct our plasmid.

According to our experiment results to judge, the ple coding sequence is hard to clone from Drosophila 's cDNA library because of its multi-segment repeats. So we recommend that you obtain from the constructed plasmid, or synthesize it directly. We ordered a synthetic ple from GENEWIZ®, and cloned it into pUAST vector with two restriction sites: EcoRI and XbaI.

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]

We cloned UAS-TH into shipping backbone pSB1C3 with In-Fusion. Here shows the restriction endonuclease digestion image of pSB1C3-UAS-TH.

[http://2017.igem.org/Team:Tongji_China/Design More Information]

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 137
Illegal XhoI site found at 676
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 595
Illegal BsaI.rc site found at 2271
Illegal SapI site found at 960

@@ Line 4: / Line 4: @@
 The expression of GAL4 is controlled by TH(ple) promoter.
+===Design notes===
+'''ple''' has three alternatively spliced transcript variants which encode iosforms of TH. We choose isoform B to construct our plasmid.
+[[File:2017tongji_image_registry_TH_base.png|center|300px|标题]]
+According to our experiment results to judge, the '''ple''' coding sequence is hard to clone from ''Drosophila'' 's cDNA library because of its multi-segment repeats.
+So we recommend that you obtain from the constructed plasmid, or synthesize it directly.
+We ordered a synthetic '''ple''' from GENEWIZ®, and cloned it into pUAST vector with two restriction sites: EcoRI and XbaI.
+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.
+[[File:2017tongji_image_registry_ple4.png|center|200px|pleP-GAL4]]
+[[File:2017tongji_image_registry_ple80.png|center|200px|pleP-GAL80ts]]
+[[File:2017tongji_image_registry_uTH.png|center|200px|pleP-GAL80ts]]
+'''''note''''':
+. 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>
+. 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]
+We cloned UAS-TH into shipping backbone pSB1C3 with In-Fusion. Here shows the restriction endonuclease digestion image of pSB1C3-UAS-TH.
+[[File:2017tongji image registry 4test.png|center|400px|标题]]
+[http://2017.igem.org/Team:Tongji_China/Design More Information]
 <!-- Add more about the biology of this part here