Team:Tongji China/Parts


Tongji iGEM - Parts
Tongji iGEM
TongJi iGEM
Parts
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Introduction

Our plasmids are based on pUAST+3xHA, for which works for UAS-GAL4 system in Drosophila, and we transform it to pUAST+oligo which removed UAS and inserted a new MCS for constructing needs.


pUAST-3xHA is an expression vector commonly used in Drosophila research, and it is based on pUC8 vector.
The P3 and P5 are the integrated efficiency elements from viruses, which lead the parts we constructed integrate to the Drosophila’s genome.
Hsp70 TATA is the site of contact for RNA polymerase II, in which will start the transcription of downstream gene inserted into MCS.
SV40 will start the expression of gene mini white, which could change the eyes’ color into red (white in wild type) when we integrate successfully. So it can help us screen out the modified flies.

Basic Parts

Name Type Description Length
BBa_K2374001 Regulatory TH (ple) promoter-> (fruit fly) 458
BBa_K2374002 Coding GAL80ts (temperature dependent) 1317
BBa_K2374003 Coding ple (Tyrosine 3-monooxygenase, TH) -> (fruit fly) 1881
BBa_K2374004 Coding GAL4 (GALactose-responsive transcription factor) 2652
BBa_K2374008 DNA UAS 375

BBa_K2374001—TH (ple) promoter-> (fruit fly)
Pale is a tyrosine hydroxylase, the first and rate-limiting step in the synthesis of dopamine (and eventually, melanin). Dopamine has critical roles in system development. This part is a 452bp sequence in 5' upstream of the ple exon. This promoter starts in cell which express dopamine specifically. So we use it to start the expression of downstream gene GAL4 and GAL80ts in tissue which express TH.

BBa_K2374002—GAL80ts (temperature dependent)
GAL80ts is a mutation based on GAL80 which is a transcription regulator in Saccharomyces cerevisiae S288C. The activity of GAL80ts is temperature dependent. At 25℃, it is activated and will bind to GAL4 then deactivate it. At 29℃, GAL80ts were inactivated and could not bind to GAL4. For the temperature between 25-29℃, we did not detect.

BBa_K2374003—ple (Tyrosine 3-monooxygenase, TH) -> (fruit fly)
ple (TH) is a rate-limiting enzyme in the dopamine’s synthesis, and it plays an important role in the physiology of adrenergic neurons.
Catalytic activity:
L-tyrosine + tetrahydrobiopterin + O2 = L-dopa + 4a-hydroxytetrahydrobiopterin. This subpathway is part of the pathway dopamine biosynthesis, which is itself part of catecholamine biosynthesis.
NO DESCRIPTION

BBa_K2374004—GAL4 (GALactose-responsive transcription factor)
In yeast, GAL4 regulates the GALactose metabolism. Transcriptional activation of GALactose utilization genes occurs when 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. The N-terminal region mediates both dimerization and DNA binding and contains a nuclear localization signal, while an acidic C-terminal domain controls transcriptional activation. GAL4 directly interacts with the Tra1 component of the SAGA complex, recruiting Mediator and the general transcriptional machinery to initiate transcription. This ability to activate transcription is retained when GAL4 is expressed in other species including plants, human cell lines, zebrafish, and Drosophila.

BBa_K2374008—UAS
An upstream activating sequence or upstream activation sequence (UAS) is a cis-acting regulatory sequence. It is distinct from the promoter and increases the expression of a neighboring gene. Due to its essential role in activating transcription, the UAS is often considered to be analogous to the function of the enhancer in multicellular eukaryotes. Upstream activation sequences are a crucial part of induction, enhancing the expression of the protein of interest through increased transcriptional activity. The UAS is found adjacently upstream to a minimal promoter (TATA box) and serves as a binding site for transactivators. If the transcriptional transactivator does not bind to the UAS in the proper orientation then transcription cannot begin.

Composite Parts

Name Type Description Length

BBa_K2374005

Composite

TH-GAL4

3110

BBa_K2374006

Composite

TH-GAL80ts

1777

BBa_K2374007

Composite

UAS-TH -> (fruit fly)

2264


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.

BBa_K2374006—TH-GAL80ts
A dimer of GAL80 binds to the C-terminal ends of the GAL4 dimer so that, while it can still bind to a UAS sequence, it can no longer activate transcription. This interaction of GAL4 and GAL80 can be taken advantage of to refine the expression pattern of GAL4-dependent transgenes.
We use the specific promoter pleP (TH Promoter) to control the fixed expression of GAL80ts, because of the specificity of pleP, GAL80ts express in tissue which express dopamine specifically. At 25℃, GAL4 and GAL80ts express, GAL80tsp conbine with GAL4p then stop it to bind to UAS, so the TH do not express.At 29℃, GAL80ts is inactivated, which cannot combine with GAL4p, so GAL4p binds to UAS and starts the expression of TH, leading to the high expression of dopamine.

BBa_K2374007—UAS-TH -> (fruit fly)
The expression level of TH is controlled by upstream activating sequence, when GAL4 binds to the GAL4 binding sequence, RNA polymerase II will bind to the downstream hsp70 TATA and start the expression of neighboring gene TH, in which could overexpress it, and then increase the expression of dopamine.

References

1. Webster Nocholas, Jin Jiarui, Green Stephen, Hollis Melvyn, Chambon Pierre. The Yeast UASG is a transcriptional enhancer in human hela cells in the presence of the GAL4 trans-activator. Cell. 1988; 52 (2): 169–178.
2. West Jr. Robert W., Yocum R. Rogers, Ptashne Mark. Saccharomyces cerevisiae GAL1-GAL10 Divergenet Promoter Region: Location and Function of the Upstream Activating Sequence UAS. Molecular and Cellular Biology. 1984; 4 (11): 2467–2478.
3. Lewandoski Mark. Conditional control of gene expression in the mouse. Nature Reviews Genetics. 2001; 2: 743–755.
4.Harrington CA, Lewis EJ, Krzemien D, Chikaraishi DM. Identification and cell type specificity of the tyrosine hydroxylase gene promoter. Nucleic Acids Research. 1987;15(5):2363-2384.
Ignis Fly

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