Difference between revisions of "Part:BBa K4890015"
 
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We also reformed the pUAST plasmid into pMRE plasmid by restrictive endonuclease (Pst1) digestion to obtain a linearized pUAST vector and then substitute UAS sequence for MRE sequence. MRE was obtained by DNA synthesis and T4 ligase was used to combine the linearized vector into complete plasmid. pMRE plasmid was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (600 bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmids extracted from the colonies were confirmed to be pMRE by gene sequencing.
 
We also reformed the pUAST plasmid into pMRE plasmid by restrictive endonuclease (Pst1) digestion to obtain a linearized pUAST vector and then substitute UAS sequence for MRE sequence. MRE was obtained by DNA synthesis and T4 ligase was used to combine the linearized vector into complete plasmid. pMRE plasmid was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (600 bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmids extracted from the colonies were confirmed to be pMRE by gene sequencing.
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Start from pMRE as template, we used restrictive endonuclease (NotI and XbaI) digestion to obtain a linearized pMRE vector. GFP gene fragment was amplified from the plasmid of pUAST-GFP by PCR. DNA electrophoresis confirmed the length of the PCR products (720 bp). GFP gene fragment was ligated with pMRE linearized vector by T4 ligase. pMRE-GFP plasmid was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (720 bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pMRE-GFP by gene sequencing.
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<br />Start from pMRE as template, we used restrictive endonuclease (NotI and XbaI) digestion to obtain a linearized pMRE vector. GFP gene fragment was amplified from the plasmid of pUAST-GFP by PCR. DNA electrophoresis confirmed the length of the PCR products (720 bp). GFP gene fragment was ligated with pMRE linearized vector by T4 ligase. pMRE-GFP plasmid was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (720 bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pMRE-GFP by gene sequencing.
  
 
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     <img class="bild" src="https://static.igem.wiki/teams/4890/wiki/parts/14-01-15-01-gel-electrophoresis-of-mre.png">
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     <img id="Figure1" class="bild" src="https://static.igem.wiki/teams/4890/wiki/parts/14-01-15-01-gel-electrophoresis-of-mre.png">
 
     <div class="unterschrift"><b>Figure 1 Gel electrophoresis of MRE</b>
 
     <div class="unterschrift"><b>Figure 1 Gel electrophoresis of MRE</b>
 
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     <div class="unterschrift"><b>Figure 2 Gel electrophoresis of GFP</b>
 
     <div class="unterschrift"><b>Figure 2 Gel electrophoresis of GFP</b>
 
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     <div class="unterschrift"><b>Figure 3 Gene sequencing of pMRE</b>
 
     <div class="unterschrift"><b>Figure 3 Gene sequencing of pMRE</b>
 
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     <div class="unterschrift"><b>Figure 4 Gel electrophoresis of pMRE-GFP plasmid (From left to right: marker, pUAST-MTF-1, pMRE-Hid and pMRE-GFP)</b>
 
     <div class="unterschrift"><b>Figure 4 Gel electrophoresis of pMRE-GFP plasmid (From left to right: marker, pUAST-MTF-1, pMRE-Hid and pMRE-GFP)</b>
 
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<h2>2 Generation of Drosophila line with genotype of UAS-MTF-1; MRE-GFP/GMR-GAL4</h2>
 
<h2>2 Generation of Drosophila line with genotype of UAS-MTF-1; MRE-GFP/GMR-GAL4</h2>
  
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<h5>(2.1) Construction of Drosophila lines</h5>
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<h5>2.1 Construction of Drosophila lines</h5>
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pUAST-MTF-1 and pMRE-GFP were micro-injected into the embryos of Drosophila W1118 respectively to obtain Drosophila UAS-MTF-1 and Drosophila MRE-GFP  (Micro-injection was performed by Core Facility of Drosophila Resource and Technology, CEMCS, CAS) . Drosophila UAS-MTF-1 was crossed with Drosophila MRE-GFP to obtain the offspring with genotype of UAS-MTF-1; MRE-GFP. This progeny was crossed with Drosophila GMR-GAL4 to obtain the progeny with genotype UAS-MTF-1;MRE-GFP/GMR-GAL4.
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Drosophila UAS-MTF-1;MRE-GFP/GMR-GAL4 was divided into 3 groups. The control group received no treatment, and the other 2 groups were treated with 10μM CdCl2, and 100μM CdCl2, respectively. 
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<h5>(2.2) Heavy metal response of Drosophila larvae: fluorescence intensity</h5>
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pUAST-MTF-1 and pMRE-GFP were micro-injected into the embryos of Drosophila W<sup>1118</sup> respectively to obtain Drosophila UAS-MTF-1 and Drosophila MRE-GFP  (Micro-injection was performed by Core Facility of Drosophila Resource and Technology, CEMCS, CAS) . Drosophila UAS-MTF-1 was crossed with Drosophila MRE-GFP to obtain the offspring with genotype of UAS-MTF-1; MRE-GFP. This progeny was crossed with Drosophila GMR-GAL4 to obtain the progeny with genotype UAS-MTF-1;MRE-GFP/GMR-GAL4.
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<br />Drosophila UAS-MTF-1;MRE-GFP/GMR-GAL4 was divided into 3 groups. The control group received no treatment, and the other 2 groups were treated with 10μM CdCl<sub>2</sub>, and 100μM CdCl<sub>2</sub>, respectively. 
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<br /><h5>2.2 Heavy metal response of Drosophila larvae: fluorescence intensity</h5>
  
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The 3rd instar larvae were collected in about 5 days and imaginal discs were dissected. The eye imaginal discs of Drosophila UAS-MTF-1;MRE-GFP/GMR-GAL4 cultured under 10μM and 100μM CdCl2 had higher fluorescence intensity than the control (P<0.05, Figure 5). The fluorescence intensity was dependent on the concentration of CdCl2.
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The third instar larvae were collected in about 5 days and imaginal discs were dissected. The eye imaginal discs of Drosophila UAS-MTF-1;MRE-GFP/GMR-GAL4 cultured under 10μM and 100μM CdCl<sub>2</sub> had higher fluorescence intensity than the control (P<0.05, Figure 5). The fluorescence intensity was dependent on the concentration of CdCl<sub>2</sub>.
  
 
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     <div class="unterschrift"><b>Figure 5 Fluorescence intensity in the eye imaginal discs of Drosophila UAS-MTF-1;MRE-GFP/GMR-GAL4 cultured under CdCl<sub>2</sub> for 5 days (unpaired t-test (vs control): ****P<0.0001)
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UAS-MTF-1;MRE-GFP/GMR-GAL4 cultured under CdCl2 for 5 days (unpaired t-test (vs control): ****P<0.0001)
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Latest revision as of 07:14, 9 October 2023


MRE-Hsp70-GFP

This part is responsible to the expression of GFP gene driven by MRE in Drosophila. It consists of MRE sequence (BBa_K4890002), Hsp70 sequence (BBa_K4890004) and GFP gene (BBa_S03452). Metal response element (MRE) is derived from Drosophila melanogaster. In the cell nucleus, activated MTF-1 binds to MRE. MRE is typically located in the promoter regions associated with genes involved in the response to heavy metals. Binding of MTF-1 to MRE activates the transcription of the corresponding genes, thereby promoting heavy metal-related gene expression. GFP gene is derived from plasmid pcDNA3.1-CMV.GFP which was kept by Genetic Lab, School of Life science and Technology, Tongji University. It encodes green fluorescent protein (GFP) that emits green fluorescence when exposed to light in the blue to ultraviolet range. Hsp70 is derived from pUAST plasmid. Hsp70 is a promoter that can bind to RNA polymerase and start transcription.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 196
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 196
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 196
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 196
  • 1000
    COMPATIBLE WITH RFC[1000]


Results

1 Construction of pMRE-GFP plasmid

We also reformed the pUAST plasmid into pMRE plasmid by restrictive endonuclease (Pst1) digestion to obtain a linearized pUAST vector and then substitute UAS sequence for MRE sequence. MRE was obtained by DNA synthesis and T4 ligase was used to combine the linearized vector into complete plasmid. pMRE plasmid was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (600 bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmids extracted from the colonies were confirmed to be pMRE by gene sequencing.

Start from pMRE as template, we used restrictive endonuclease (NotI and XbaI) digestion to obtain a linearized pMRE vector. GFP gene fragment was amplified from the plasmid of pUAST-GFP by PCR. DNA electrophoresis confirmed the length of the PCR products (720 bp). GFP gene fragment was ligated with pMRE linearized vector by T4 ligase. pMRE-GFP plasmid was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (720 bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pMRE-GFP by gene sequencing.

Figure 1 Gel electrophoresis of MRE

Figure 2 Gel electrophoresis of GFP

Figure 3 Gene sequencing of pMRE

Figure 4 Gel electrophoresis of pMRE-GFP plasmid (From left to right: marker, pUAST-MTF-1, pMRE-Hid and pMRE-GFP)

2 Generation of Drosophila line with genotype of UAS-MTF-1; MRE-GFP/GMR-GAL4

2.1 Construction of Drosophila lines
pUAST-MTF-1 and pMRE-GFP were micro-injected into the embryos of Drosophila W1118 respectively to obtain Drosophila UAS-MTF-1 and Drosophila MRE-GFP (Micro-injection was performed by Core Facility of Drosophila Resource and Technology, CEMCS, CAS) . Drosophila UAS-MTF-1 was crossed with Drosophila MRE-GFP to obtain the offspring with genotype of UAS-MTF-1; MRE-GFP. This progeny was crossed with Drosophila GMR-GAL4 to obtain the progeny with genotype UAS-MTF-1;MRE-GFP/GMR-GAL4.

Drosophila UAS-MTF-1;MRE-GFP/GMR-GAL4 was divided into 3 groups. The control group received no treatment, and the other 2 groups were treated with 10μM CdCl2, and 100μM CdCl2, respectively.

2.2 Heavy metal response of Drosophila larvae: fluorescence intensity
The third instar larvae were collected in about 5 days and imaginal discs were dissected. The eye imaginal discs of Drosophila UAS-MTF-1;MRE-GFP/GMR-GAL4 cultured under 10μM and 100μM CdCl2 had higher fluorescence intensity than the control (P<0.05, Figure 5). The fluorescence intensity was dependent on the concentration of CdCl2.

Figure 5 Fluorescence intensity in the eye imaginal discs of Drosophila UAS-MTF-1;MRE-GFP/GMR-GAL4 cultured under CdCl2 for 5 days (unpaired t-test (vs control): ****P<0.0001)