Part:BBa_K4890014

MRE-Hsp70-Hid

This part is responsible to the expression of Hid gene driven by MRE in Drosophila. It consists of MRE sequence (BBa_K4890002), Hsp70 sequence (BBa_K4890004) and Hid gene (BBa_K4890003). Metal response element (MRE) is derived from Drosophila melanogaster. In the cell nucleus, activated MTF-1 binds to MRE. The 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. Head involution defective (Hid) gene is derived from Drosophila melanogaster. Hid induces cell apoptosis in Drosophila. 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 EcoRI site found at 360
  Illegal XbaI site found at 1616
  Illegal PstI site found at 222
  Illegal PstI site found at 964
  Illegal PstI site found at 1193
  Illegal PstI site found at 1337
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 360
  Illegal PstI site found at 222
  Illegal PstI site found at 964
  Illegal PstI site found at 1193
  Illegal PstI site found at 1337
  Illegal NotI site found at 378
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 360
  Illegal BglII site found at 372
  Illegal XhoI site found at 487
  Illegal XhoI site found at 1393
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 360
  Illegal XbaI site found at 1616
  Illegal PstI site found at 222
  Illegal PstI site found at 964
  Illegal PstI site found at 1193
  Illegal PstI site found at 1337
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 360
  Illegal XbaI site found at 1616
  Illegal PstI site found at 222
  Illegal PstI site found at 964
  Illegal PstI site found at 1193
  Illegal PstI site found at 1337
  Illegal NgoMIV site found at 1322
  
• 1000
  COMPATIBLE WITH RFC[1000]

1 Construction of pMRE-Hid 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. Plasmid extracted from the colonies was 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. Hid gene fragment was amplified from the cDNA of wildtype Drosophila melanogaster by PCR. DNA electrophoresis confirmed the length of the PCR product (1233bp). Hid gene fragment was ligated with pMRE linearized vector by T4 ligase. pMRE-Hid plasmid was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (1233bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pMRE-Hid by gene sequencing.

Figure 1 Gel electrophoresis of MRE

Figure 2 Gel electrophoresis of Hid

Figure 3 Gene sequencing of pMRE

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

We cultured Drosophila S2 cells on plates for 24h, and then transiently co-transfected pUAST-MTF-1 (refer to BBa_K4890013), pMRE-Hid and pAc-GAL4 (which contains actin-GAL4) plasmids into the S2 cells. pAc-GAL4 plasmid was previous constructed by Genetic Lab, School of Life Science and Technology, Tongji University. The transfected S2 cells were notated as Drosophila UAS-MTF-1/MRE-Hid/Ac-GAL4 cells.

Drosophila UAS-MTF-1/MRE-Hid/Ac-GAL4 cells were cultured for 48h and then divided into 5 groups. The control group received no treatment, and the other 4 groups were treated with 10μM ZnCl2, 100μM ZnCl2, 10μM CdCl2, and 100μM CdCl2, respectively, for 4h.

Real-time PCR and Western Blot results confirmed that both mRNAs and proteins of MTF-1 and Hid were expressed in Drosophila UAS-MTF-1/MRE-Hid/Ac-GAL4 cells treated with 10μM and 100μM ZnCl2 or CdCl2 (Figure 5-6). The mRNA and protein levels of Hid were concentration-dependent (P<0.05). The mRNA level of MTF-1 was not changed with the addition of metal ions (P>0.05).

Figure 5 mRNA levels of MTF-1 and Hid in Drosophila UAS-MTF-1/MRE-Hid/Ac-GAL4 cells treated with different concentrations of ZnCl2 or CdCl2 (unpaired t-test: ***P<0.001, ****P<0.0001, ns: P>0.05)