Difference between revisions of "Part:BBa K2374002"
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<partinfo>BBa_K2374002 short</partinfo>
 
<partinfo>BBa_K2374002 short</partinfo>
  
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Gal80ts is a mutation based on Gal80 which is a transcription regulator in ''Saccharomyces cerevisiae'' S288C.
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===Overview===
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The activity of Gal80ts is temperature dependent. At 25&#8451;, it has a normal activity, which will bind to Gal4 and deactivate it. At 29&#8451;, Gal80ts were inactivated and could not bind to Gal4. For the temperature between 25-29&#8451;, we did not detect.
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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.<br>
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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.
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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.  
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===Design notes===
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[[File:2017tongji_image_registry_ple4.png|right|200px|pleP-GAL4]] 
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[[File:2017tongji_image_registry_ple80.png|right|200px|pleP-GAL80ts]] 
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[[File:2017tongji_image_registry_uTH.png|right|200px|pleP-GAL80ts]] 
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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.
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===Test Results===
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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>
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[[File:2017tongji_image_registry_qPCR.png|center|300px]]
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2. Detect male-male courtship when raising the temperature. Mating index refers to the relative time that the fruit fly use for mating.<br>
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It shows that he mating index of the treated group rises significantly in at 29°C. [time=5minutes, n=5, P<0.01]
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[[File:2017tongji_image_registry_behavior1.png|center|350px]]
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[http://2017.igem.org/Team:Tongji_China/Experiments More details]
  
  
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===Usage and Biology===
 
===Usage and Biology===
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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.
  
 
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===Functional Parameters===
 
===Functional Parameters===
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GAL80ts represses GAL4 by physical interaction between 18°C to 29°C.
 
<partinfo>BBa_K2374002 parameters</partinfo>
 
<partinfo>BBa_K2374002 parameters</partinfo>
 
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Revision as of 20:25, 30 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

pleP-GAL4
pleP-GAL80ts
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 (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.


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.

2017tongji image registry qPCR.png

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]

2017tongji image registry behavior1.png

[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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 993
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 19
    Illegal BglII site found at 615
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 27
    Illegal BsaI site found at 73


Functional Parameters

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