1 Abstract
Our team aims to develop a convenient and inexpensive visual monitoring system for heavy metals by constructing transgenic Drosophila. We inserted MTF-1 gene driven by UAS and Hid gene driven by MRE in Drosophila S2 cells, respectively. These two transgenic Drosophila lines interbreed and the offspring with both MTF-1 and Hid genes cross with the Drosophila line with genotype GMR-GAL4 or Vg-GAL4 or ptc-GAL4 which express GAL4 during eye/wing development. In this way, we constructed three Drosophila lines with genotype UAS-MTF-1;MRE-Hid/GMR-GAL4, UAS-MTF-1;MRE-Hid/Vg-GAL4, and UAS-MTF-1;MRE-Hid/ptc-GAL4 which express MTF-1 during eye/wing development due to the GAL4-UAS system. In response to heavy metals in the environment, MTF-1 is activated, binds to MRE and activates Hid expression, leading to apoptosis in the developing eyes/wings. Thus, in the first generation within 5-10 days, these lines show abnormally small eye/wing sizes observable by naked eyes. The system is sensitive, cost-effective, and environmentally friendly, requiring minimum equipment and professional personnel, and is especially of great value to remote and underdeveloped regions.
Our team successfully constructed 9 parts. BBa_K4890001, BBa_K4890004, BBa_K4890011, and BBa_K4890013 were developed for expressing MTF-1 gene driven by UAS. BBa_K4890002- BBa_K4890004, BBa_K4890012 and BBa_K4890014 were developed for expressing Hid gene driven by MRE. Plasmids based on these parts and pAc-GAL4 plasmid were co-transfected into Drosophila S2 cells to verified the efficiency of this monitor system in response to heavy metals. The cells was assessed by PCR, gene sequencing, real-time PCR and Western blot. Engineering based on these plasmids and hybridization were used to construct Drosophila lines with genotype UAS-MTF-1;MRE-Hid/GMR-GAL4, UAS-MTF-1;MRE-Hid/Vg-GAL4, and UAS-MTF-1;MRE-Hid/ptc-GAL4. Treated with heavy metal ions, eye/wing imaginal discs from larvae were evaluated by AO staining and Dcp-1 staining and their adults’ eye/wing areas were analyzed. BBa_K4890015 was developed for expressing GFP gene driven by MRE. Engineering based on this part and hybridization were used to construct Drosophila line with genotype UAS-MTF-1;MRE-GFP/GMR-GAL4. Treated with heavy metal ions, fluorescence intensity in the eye imaginal discs from larvae was analyzed.
| No. | Part Number | Type | Description | Designer | Length |
| 1 | BBa_K4890001 | Basic | MTF-1 | 2535 | |
| 2 | BBa_K4890002 | Basic | MRE | 73 | |
| 3 | BBa_K4890003 | Basic | Hid | 1537 | |
| 4 | BBa_K4890004 | Basic | Hsp70 | 262 | |
| 5 | BBa_K4890011 | Composite | UAS-Hsp70 | 342 | |
| 6 | BBa_K4890012 | Composite | MRE-Hsp70 | 320 | |
| 7 | BBa_K4890013 | Composite | UAS-Hsp70-MTF1 | 2724 | |
| 8 | BBa_K4890014 | Composite | MRE-Hsp70-Hid | 1556 | |
| 9 | BBa_K4890015 | Composite | MRE-Hsp70-GFP | 1032 |
2 Missions accomplished
To construct Drosophila lines with genotype of UAS-MTF-1;MRE-Hid/GMR-GAL4, UAS-MTF-1;MRE-Hid/Vg-GAL4, and UAS-MTF-1;MRE-Hid/ptc-GAL4, we accomplished three missions.
2.1 Build
Mission 1: Construction of pUAST-MTF-1, pMRE-Hid, and pMRE-GFP plasmids (1)Construction of pUAST-MTF-1 plasmidWe took a commercialized recombinant plasmid pUAST as template, and used restrictive endonuclease (BglII and XhoI) digestion to obtain a linearized pUAST vector. MTF-1 gene fragment was amplified from the cDNA of wildtype Drosophila melanogaster by PCR. DNA electrophoresis confirmed the length of the PCR product (2376bp). MTF-1 gene fragment was ligated with the pUAST linearized vector by T4 ligase. pUAST-MTF-1 was transformed into E. coli DH5α strain. Colony PCR and DNA electrophoresis (2376bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmid extracted from the colonies was confirmed to be pUAST-MTF-1 plasmid by gene sequencing (Figure 1-2,4).
(2) Construction of pMRE-Hid and pMRE-GFP plasmidsWe 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 (Figure 2-3).
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. GFP gene fragment was amplified from the plasmid of pUAST-GFP by PCR. DNA electrophoresis confirmed the lengths of the PCR products (1233bp and 720bp). Hid and GFP gene fragments were ligated with pMRE linearized vector by T4 ligase, respectively. pMRE-Hid and pMRE-GFP plasmids were transformed into E. coli DH5α strain, respectively. Colony PCR and DNA electrophoresis (1233bp and 720 bp) was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmids extracted from the colonies were confirmed to be pMRE-Hid and pMRE-GFP by gene sequencing (Figure 1-2, 4).
Fig.1 Genetic circuits of pUAST-MTF-1, pMRE-Hid, and pMRE-GFP plasmids
Figure 2 Gel electrophoresis of PCR products for the construction of pUAST-MTF-1, pMRE-Hid, and pMRE-GFP plasmids
Figure 3 Gene sequencing of pMRE
Figure 4 Gel electrophoresis of pUAST-MTF-1, pMRE-Hid, and pMRE-GFP plasmids (From left to right: marker, pUAST-MTF-1, pMRE-Hid and pMRE-GFP)
2.2 Test
Mission 2: Transient co-transfection of Drosophila S2 cells with pUAST-MTF-1, pMRE-Hid, and pAc-GAL4 plasmidsWe cultured Drosophila S2 cells on plates for 24h, and then transiently co-transfected pUAST-MTF-1, pMRE-Hid and pAc-GAL4 (which contains actin-GAL4) plasmids into the S2 cells by using ROCHE X-treme GENE™ HP DNA Transfection Reagent. 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).
unpaired t-test: ***P<0.001,****P<0.0001, ns: 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
Note: MTF-1 was detected by antibody of HA tag, which was co-expressed at the N-terminal of MTF-1, Hid was detected by antibody of Myc tag, which was co-expressed at the C-terminal of Hid
Figure 6 Protein levels of MTF-1 and Hid in Drosophila UAS-MTF-1/MRE-Hid/Ac-GAL4 cells treated with different concentrations of ZnCl2 or CdCl2
Mission 3: Generation of Drosophila lines with genotype of UAS-MTF-1;MRE-Hid/GMR-GAL4, UAS-MTF-1;MRE-Hid/Vg-GAL4, UAS-MTF-1;MRE-Hid/ptc-GAL4, and UAS-MTF-1;MRE-GFP/GMR-GAL4(1) Construction of Drosophila lines
pUAST-MTF-1, pMRE-Hid and pMRE-GFP were micro-injected into the embryos of Drosophila W1118 respectively to obtain Drosophila UAS-MTF-1, Drosophila MRE-Hid 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-Hid to obtain the offspring with genotype of UAS-MTF-1;MRE-Hid. This progeny was crossed with Drosophila GMR-GAL4 or Drosophila Vg-GAL4 or Drosophila ptc-GAL4 to obtain the progeny with genotype UAS-MTF-1;MRE-Hid/GMR-GAL4 or UAS-MTF-1;MRE-Hid/Vg-GAL4 or UAS-MTF-1;MRE-Hid/ptc-GAL4.
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
(2) Group assignment of Drosophila lines
Each cell line of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 and Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4 was divided into 5 groups for larvae Acridine Orange (AO) staining and adult phenotype. The control group received no treatment, and the other 4 groups were treated with 10μM ZnCl2, 100μM ZnCl2, 10μM CdCl2, and 100μM CdCl2, respectively.
Each cell line of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 and Drosophila UAS-MTF-1;MRE-Hid/ptc-GAL4 were divided into 3 groups for larvae Death Caspase-1 (Dcp-1) staining. The control group received no treatment, and the other 2 groups were treated with 10μM CdCl2, and 100μM CdCl2, respectively.
Drosophila UAS-MTF-1;MRE-GFP/GMR-GAL4 line was divided into 3 groups for detection of fluorescence intensity. The control group received no treatment, and the other 2 groups were treated with 10μM CdCl2, and 100μM CdCl2, respectively.
For larvae AO staining and Dcp-1 staining, the 3rd instar larvae were collected in about 5 days and imaginal discs were dissected.
(3) Heavy metal response of Drosophila larvae: cell death
In Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4, AO staining detected increased cell death in the eye imaginal discs when cultured with ZnCl2 (100μM) or CdCl2 (10μM and 100μM) (P<0.01) (Figure 7). The level of cell death was correlated with the concentration of CdCl2 (P<0.05). More cell death was observed in Drosophila grown under 10μM and 100μM CdCl2 than in those under the same concentration of ZnCl2 (P<0.05)
In Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4, AO staining detected increased cell death in the wing imaginal discs when cultured with 10μM and 100μM ZnCl2 or CdCl2 (P<0.05) (Figure 8). The level of cell death was correlated with the concentration of metal ions (P<0.05). More cell death was observed in Drosophila grown under 10μM and 100μM CdCl2 than in those under the same concentration of ZnCl2(P<0.05).
unpaired t-test (vs control): **P<0.01, ***P<0.001, ****P<0.0001, ns: P>0.05
Figure 7 Cell death in the eye imaginal discs of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 cultured under ZnCl2 and CdCl2 for 5 days detected by AO staining
unpaired t-test (vs control): *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001
Figure 8 Cell death in the wing imaginal discs of Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4 cultured under ZnCl2 and CdCl2 for 5 days detected by AO staining
(4) Heavy metal response of Drosophila larvae: cell apoptosis
Dcp-1 staining detected enhanced cell apoptosis in the eye or wing imaginal discs of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 and Drosophila UAS-MTF-1;MRE-Hid/ptc-GAL4 cultured under 100μM CdCl2 (P<0.05)(Figure 9-10). Cultured under 10μM CdCl2, their discs also showed increased trend of cell apoptosis, but the difference was not statistically significant compared with the control (P>0.05).
unpaired t-test (vs control): ***P<0.001, ns: P>0.05
Figure 9 Cell apoptosis in the eye imaginal discs of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 cultured under CdCl2 for 5 days detected by Dcp-1 staining
unpaired t-test (vs control): ****P<0.0001, ns: P>0.05
Figure 10 Cell apoptosis in the wing imaginal discs of Drosophila UAS-MTF-1;MRE-Hid/ptc-GAL4 cultured under CdCl2 for 5 days detected by Dcp-1 staining
(5) Heavy metal response of Drosophila larvae: fluorescence intensity
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 11). The fluorescence intensity was dependent on the concentration of CdCl2
unpaired t-test (vs control): ****P<0.0001
Figure 11 Fluorescence intensity in the eye imaginal discs of Drosophila UAS-MTF-1;MRE-GFP/GMR-GAL4 cultured under CdCl2 for 5 days
(6) Heavy metal response of Drosophila adults: eye/wing area
The F1 generation adults were collected in about 10 days.
Eye areas of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 adults cultured under 10μM and 100μM ZnCl2 or CdCl2 were all smaller than the control (P<0.05) (Figure 12). With the increase of metal ion concentration, the eye area decreased. Eye areas were more sensitive to CdCl2 than ZnCl2.
Wing areas of Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4 adults showed the similar results (Figure 13).
unpaired t-test (vs control): ***P<0.001, ****P<0.0001
Figure 12 Eye areas of Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 adults cultured under ZnCl2 and CdCl2 for 10 days
unpaired t-test (vs control): ****P<0.0001
Figure 13 Wing areas of Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4 adults cultured under ZnCl2 and CdCl2 for 10 days
3 Response modeling to heavy matals
4 Conclusion and Future work
This project involves the creation of a heavy metal pollution monitoring system that utilizes genetically modified Drosophila. The system is designed to visually determine whether a sample contains high concentrations of heavy metals by observing changes in the sizes of the Drosophila's eyes and wings. The process involves rearing gene-edited adult Drosophila on culture medium containing food, soil, or water samples in tubes for 5-10 days. If the samples contain high levels of heavy metals, the offspring Drosophila's eye and wing cells will undergo apoptosis, leading to a reduction in eye and wing areas.
The primary advantage of this method is cost-effectiveness. Drosophila is relatively easy to cultivate and maintain, require minimal resources, and do not necessitate expensive equipment. Furthermore, the method is user-friendly. Given their small size and short monitoring period, Drosophila can be easily transported to remote areas for long-term monitoring. This method is also environmentally friendly and highly sensitive.
However, there are limitations to this monitoring system. It cannot accurately quantify heavy metal contamination, identify specific types of heavy metals, or detect multiple heavy metals. Variations in sample concentrations within the Drosophila tubes may result in biased results.
In future research, we aim to develop a multi-metal monitoring technique for Drosophila and enhance their genetic makeup to exhibit distinguishable traits when exposed to multiple metals. This will enable us to monitor different types of heavy metal contamination as well as contamination involving multiple heavy metals. We also plan to refine the monitoring methods to ensure more uniform sample ingestion or exposure. Consideration is being given to incorporating sensor technology into the system for real-time monitoring of the Drosophila's status.