Difference between revisions of "Part:BBa K2374002"

Revision as of 18:24, 31 October 2017

GAL80ts (temperature dependent)

Overview

The activity of GAL4 can be repressed by physical interaction with the yeast GAL80 protein. 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.
Gal80ts is a temperature sensitive variant thought to be the result of a single glycine to arginine substitution at amino acid 203 of GAL80. It is a transcription regulator in Saccharomyces cerevisiae S288C. GAL80ts is unable to bind GAL4 at restrictive temperatures above 29 °C but retains its repressive function at the permissive temperature of 18 °C . Controlling the activity of GAL80ts through temperature shifts provides temporal control of GAL4-dependent transgene expression.

Design notes

We combined this promoter with UAS/GAL4 system to achieve gene expression in specific cells.
We cloned this 452bp TH promoter easily from D. melanogaster 's genomic DNA and the sequencing result is correct.

We connected TH promoter to GAL4( BBa_K2374004 [1] )and GAL80ts( BBa_K2374002 [2] )respectively, and cloned them into pUAST vector which had been removed the UAS sequence( BBa_K2374008 [3] ), then to microinject them into Drosophila 's eggs. Also we did microinjection with UAS-TH (BBa_K2374003 [4]). After hybridization screening, we got stable modified fruit fly strains. Finally, we did RT-PCR, qPCR and behavioral experiments to test our system. Here shows some results[http://2017.igem.org/Team:Tongji_China/Experiments].

We cloned TH promoter into pSB1C3 for submission. Here shows the 1% Agarose gel electrophoresis image.

site direct mutagenesis:
1. EcoR I (184) GAATTC->GATTTC
2. Xba I (219) TCTAGA->TGTAGA

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]

Usage and Biology

Controlling the activity of GAL80ts through temperature shifts provides temporal control of GAL4-dependent transgene expression. Limiting expression to defined temporal windows can help to define critical periods for the effects of misexpression or rescue experiments. Temperature-sensitive alleles of GAL4 itself have been generated and tested in Drosophila, however the ease of combining tub-GAL80ts with already established GAL4 drivers cause the TARGET system to be more commonly used.

Sequence and Features

Functional Parameters

GAL80ts represses GAL4 by physical interaction between 18°C to 29°C.

Reference

Kaixia Li, Boen Ma, Jianzheng Zhang, et-al. Combination of Gal80ts and Gal4 flexibly manipulates the expression levels of UAS transgenes in Drosophila[J]. Acta Entomologica Sinica ,2016,(5):481-488.