Difference between revisions of "Part:BBa K3734025"

 
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<partinfo>BBa_K3734025 short</partinfo>
 
<partinfo>BBa_K3734025 short</partinfo>
  
 
GIP is a promoter that can be induced by glucose.And miR21T is miR21's target.When miR21 combined with miR21T, it will inhibit the expression of target gene.GI is a kind of light-activated protein. If you shine blue light on it, it will change its own structure and will be able to combined with LOV. GAL4 is a kind of protein that can find and combined with UAS DNA structure domain.
 
GIP is a promoter that can be induced by glucose.And miR21T is miR21's target.When miR21 combined with miR21T, it will inhibit the expression of target gene.GI is a kind of light-activated protein. If you shine blue light on it, it will change its own structure and will be able to combined with LOV. GAL4 is a kind of protein that can find and combined with UAS DNA structure domain.
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<h2 class="pageContent-main__title">
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                GIP-miR21T-GI-GAL4-4XmiR21T
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            <div class="pageContent-main__textBox">
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                <p>GIP-miR21T-GI-GAL4-4XmiR21T consists of glucose-induced promoter GIP, photosensitive protein GI, double yeast hybrid system components GAL4 and miR21T. It is regulated by both glucose concentration and blue <br>light, which improves the safety of the whole system. At the same time, it is also suppressed by miR21 feedback, which makes the whole system form a closed loop <br>and makes the reaction more sensitive.
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                </p>
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                <p>In order to increase the safety of patients' use, it may be difficult to achieve the expected goal of accurate regulation by relying on the regulation of blood glucose <br>concentration alone, so we designed a pair of photosensitive proteins <br>of GI (BBa_K3734004) and LOV (BBa_K3734006). Under blue light irradiation, the Protein structure changes and interacts with each other to form a GAL4-GI-LOV-VP16 quadruple, while GAL4 (BBa_K3734005) identifies and combines 9XUAS (BBa_K3734016), so that VP16 can activate the gene of downstream expression.
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                1.Pattern diagram
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                <p style="text-align: center;">
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                    <img width="400px" src="https://2021.igem.org/wiki/images/8/87/T--CSU_CHINA--tupian5.png"></p>
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                <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.1 The model diagram of GIP-miR21T-GI-GAL4-4XmiR21T</p>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                2.Experiment
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            </h2>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                2.1 Method
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                <p>We used report gene LUC to represent the effect of GIP-miR21T-GI-GAL4-4XmiR21T
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                <!--put text here, <p>content</p>-->
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                2.2 Result
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                    <img width="400px" src="https://2021.igem.org/wiki/images/3/37/T--CSU_CHINA--tupian37.png"></p>
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                <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.2 Light controlled system testing experiment</p>
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                <p style="text-align: center;">
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                    <img width="400px" src="https://2021.igem.org/wiki/images/c/cd/T--CSU_CHINA--tupian38.png"></p>
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                <p style="width: 80%;text-align:center;font-size: .9rem; margin: -1rem auto 1rem auto; color: #888;">Fig.3 Expression level analysis under blue light irradiation and dark treatment</p>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                3.Caution
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                <p>The duration of GI and LOV after 450nm blue light exposure will vary with the irradiation method, intensity, etc. In the experiment, we adopted the method of irradiating strong light for 30 minutes first, and then low light for 30 minutes.
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                    The GAL4 binding domain is universal in eukaryotic cells, but transcription activators (such as VP16) that cooperate with GAL4 need to be properly selected according to different chassis organisms.
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                </p>
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            <h2 class="pageContent-main__title pageContent-main__subtitle">
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                Reference：
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                <p>[1]Chun Jeih Ryu , Charles E Whitehurst, Jianzhu Chen.Expression of Gal4-VP16 and Gal4-DNA binding domain under the control of the T lymphocyte-specific lck proximal promoter in transgenic mice[J].BMB Rep. 2008 Aug 31;41(8):575-80.
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                </p>
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                <p>[2]Masayuki Yazawa , Amir M Sadaghiani, Brian Hsueh.Induction of protein-protein interactions in live cells using light[J].Nat Biotechnol. 2009 Oct;27(10):941-5.
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                </p>
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                <p>[3]Kai Zhang , Xue-Jiao Yang , Ting-Ting Zhang.In situ imaging and interfering Dicer-mediated cleavage process via a versatile molecular beacon probe.Anal ChimActa.2019Nov4;1079:146-152.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 13:59, 20 October 2021

GIP-miR21T-GI-GAL4-4XmiR21T

GIP is a promoter that can be induced by glucose.And miR21T is miR21's target.When miR21 combined with miR21T, it will inhibit the expression of target gene.GI is a kind of light-activated protein. If you shine blue light on it, it will change its own structure and will be able to combined with LOV. GAL4 is a kind of protein that can find and combined with UAS DNA structure domain.

GIP-miR21T-GI-GAL4-4XmiR21T

GIP-miR21T-GI-GAL4-4XmiR21T consists of glucose-induced promoter GIP, photosensitive protein GI, double yeast hybrid system components GAL4 and miR21T. It is regulated by both glucose concentration and blue
light, which improves the safety of the whole system. At the same time, it is also suppressed by miR21 feedback, which makes the whole system form a closed loop 
and makes the reaction more sensitive.

In order to increase the safety of patients' use, it may be difficult to achieve the expected goal of accurate regulation by relying on the regulation of blood glucose 
concentration alone, so we designed a pair of photosensitive proteins 
of GI (BBa_K3734004) and LOV (BBa_K3734006). Under blue light irradiation, the Protein structure changes and interacts with each other to form a GAL4-GI-LOV-VP16 quadruple, while GAL4 (BBa_K3734005) identifies and combines 9XUAS (BBa_K3734016), so that VP16 can activate the gene of downstream expression.

1.Pattern diagram

Fig.1 The model diagram of GIP-miR21T-GI-GAL4-4XmiR21T

2.Experiment

2.1 Method

We used report gene LUC to represent the effect of GIP-miR21T-GI-GAL4-4XmiR21T

2.2 Result

Fig.2 Light controlled system testing experiment

Fig.3 Expression level analysis under blue light irradiation and dark treatment

3.Caution

The duration of GI and LOV after 450nm blue light exposure will vary with the irradiation method, intensity, etc. In the experiment, we adopted the method of irradiating strong light for 30 minutes first, and then low light for 30 minutes. The GAL4 binding domain is universal in eukaryotic cells, but transcription activators (such as VP16) that cooperate with GAL4 need to be properly selected according to different chassis organisms.

Reference：

[1]Chun Jeih Ryu , Charles E Whitehurst, Jianzhu Chen.Expression of Gal4-VP16 and Gal4-DNA binding domain under the control of the T lymphocyte-specific lck proximal promoter in transgenic mice[J].BMB Rep. 2008 Aug 31;41(8):575-80.

[2]Masayuki Yazawa , Amir M Sadaghiani, Brian Hsueh.Induction of protein-protein interactions in live cells using light[J].Nat Biotechnol. 2009 Oct;27(10):941-5.

[3]Kai Zhang , Xue-Jiao Yang , Ting-Ting Zhang.In situ imaging and interfering Dicer-mediated cleavage process via a versatile molecular beacon probe.Anal ChimActa.2019Nov4;1079:146-152.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 4405
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4300
    Illegal XhoI site found at 1953
    Illegal XhoI site found at 5001
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 899
    Illegal NgoMIV site found at 2455
    Illegal AgeI site found at 3682
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4920
    Illegal BsaI.rc site found at 216
    Illegal BsaI.rc site found at 3079