Part:BBa_K2374005

TH-GAL4

We use the specific promoter pleP (TH Promoter) to control the fixed expression of GAL4, because of the specificity of pleP, GAL4 express in tissue which express dopamine specifically. Then GAL4 binds to the upstream activation sequence (UAS) containing varying numbers of a 17-mer repeat. GAL4 binds to DNA as a dimer through a Zn(2)-Cys(6) zinc finger, and directly interacts with the Tra1 component of the SAGA complex, recruiting Mediator and the general transcriptional machinery to initiate transcription.

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]

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]

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]

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