Team:Hong Kong JSS/Basic Part
In this project, we designed 6 new basic parts.
The basic parts include two coding sequence for expressing metal-binding protein - Metallothionein from Corynebacterium glutamicum (CgMT) (BBa K3076100 ) and Vanadium-binding protein from Ascidia sydneiensis samea (VB) (BBa K3076200 ). We planned to express the metal-binding proteins in E. Coli and see if they could enhance the copper removal ability of the organism. Due to time and resources limits, we were only able to test the effect of CgMT. CgMT is a metal-binding protein which reported to have a high affinity of binding divalent metal ions. [1] (Fig. 1) We also found that iGEM 2016 Team Oxford reported that MT from M. tuberculosis(BBa K1980002 ) could bind to heavy metals and copper and C. glutamicum belongs to the same order with M. tuberculosis, so we expect CgMT will display similar metal binding property which suits the aim of our project.
The basic part of CgMT contains the coding sequence of CgMT obtained from NCBI (ACCESSION: KJ638906). Then, the sequence was codon-optimized for E. Coli and synthesized into pET151/TOPO expression vector. The E. Coli strain used was BL21 (DE). VB was reported to bind vanadium with a high affinity, but when excess amount of copper (II) ions are present, the copper ions will displace the vanadium ions on the protein. [2] Therefore, this indicates that VB prefers to chelate copper (II) ions and it is a plausible protein candidate for our project. The basic part contains the coding sequence of vanabin2 gene which encodes the VB protein. The part was synthesized by IDT gBlock and planned to be assembled into pET28a expression vector by Hifi assembly method. However, due to time constraints, the work is still in progress.
We designed four double-stranded (ds) DNA substrates for Lambda red recombineering to knockout four different major gene involved in the pathway of copper ion export in E. Coli. The genes planned to be knocked out are copA (BBa K3076806 ), cusA (BBa K3076400 ), cutA (BBa K3076600 ) and cusF (BBa K3076500 ) . CopA encodes a copper-translocating P-type ATPase in E.coli [3, 4] to remove copper from the intracellular space of E. Coli to periplasmic space. Meanwhile, cusA and cusF are two major components involved in the CusCFBA copper-transporting efflux system. [5] (Fig. 2) CutA is a copper binding protein found in E. Coli and reported to help to increase the copper-resistance of E. coli by channeling copper ions to efflux system. [6]
The above dsDNA substrate were desgined to have three components. There is a double terminator followed by a kanamycin resistance gene, and these two components were flanked by the homology sequence of the gene to be knocked out. When the recombination is successful, the substrate will be recombined to the homology site of the E. Coli genome and disrupt the target gene. Meanwhile, the inserted terminator can ensure the stop of transcription of the target gene and the kanamycin gene can act as a selection marker for sucessful recombination. (Fig. 3)
These substrates can be recombined into plasmid vectors for storage. When in need, we can directly use PCR to produce the substrate in a large number.
Reference:
| Name | Type | Description |
|---|---|---|
| BBa_K3076100 | Coding | Coding sequence of metallothioneins (MT) gene from Corynebacterium glutamicum (CgMT) |
| BBa_K3076200 | Coding | Coding sequence of Vanabin2 gene from Ascidia sydneiensis samea |
| BBa_K3076400 | Other | dsDNA substrate with KanR gene for cusA knockout in E. coli by Lambda Red Recombineering |
| BBa_K3076500 | Other | dsDNA substrate with KanR gene for cusF knockout in E. coli by Lambda Red Recombineering |
| BBa_K3076500 | Other | dsDNA substrate with KanR gene for cutA knockout in E. coli by Lambda Red Recombineering |
| BBa_K3076806 | Other | dsDNA substrate with KanR gene for copA knockout in E. coli by Lambda Red Recombineering |
Metal-binding proteins
The basic parts include two coding sequence for expressing metal-binding protein - Metallothionein from Corynebacterium glutamicum (CgMT) (BBa K3076100 ) and Vanadium-binding protein from Ascidia sydneiensis samea (VB) (BBa K3076200 ). We planned to express the metal-binding proteins in E. Coli and see if they could enhance the copper removal ability of the organism. Due to time and resources limits, we were only able to test the effect of CgMT. CgMT is a metal-binding protein which reported to have a high affinity of binding divalent metal ions. [1] (Fig. 1) We also found that iGEM 2016 Team Oxford reported that MT from M. tuberculosis(BBa K1980002 ) could bind to heavy metals and copper and C. glutamicum belongs to the same order with M. tuberculosis, so we expect CgMT will display similar metal binding property which suits the aim of our project.
Fig. 1 The structure of yeast MT (left) and cyanobacteria MT (right). The spheres represent the metal ions being chelated. This indicates that MT from different organism has different metal binding affinity and amount. Image adopted from wikipedia available at: https://en.wikipedia.org/wiki/Metallothionein
The basic part of CgMT contains the coding sequence of CgMT obtained from NCBI (ACCESSION: KJ638906). Then, the sequence was codon-optimized for E. Coli and synthesized into pET151/TOPO expression vector. The E. Coli strain used was BL21 (DE). VB was reported to bind vanadium with a high affinity, but when excess amount of copper (II) ions are present, the copper ions will displace the vanadium ions on the protein. [2] Therefore, this indicates that VB prefers to chelate copper (II) ions and it is a plausible protein candidate for our project. The basic part contains the coding sequence of vanabin2 gene which encodes the VB protein. The part was synthesized by IDT gBlock and planned to be assembled into pET28a expression vector by Hifi assembly method. However, due to time constraints, the work is still in progress.
dsDNA substrate for copper exporter knockout
We designed four double-stranded (ds) DNA substrates for Lambda red recombineering to knockout four different major gene involved in the pathway of copper ion export in E. Coli. The genes planned to be knocked out are copA (BBa K3076806 ), cusA (BBa K3076400 ), cutA (BBa K3076600 ) and cusF (BBa K3076500 ) . CopA encodes a copper-translocating P-type ATPase in E.coli [3, 4] to remove copper from the intracellular space of E. Coli to periplasmic space. Meanwhile, cusA and cusF are two major components involved in the CusCFBA copper-transporting efflux system. [5] (Fig. 2) CutA is a copper binding protein found in E. Coli and reported to help to increase the copper-resistance of E. coli by channeling copper ions to efflux system. [6]
Fig. 2 Copper homeostasis mechanisms in E. Coli. Adopted from Kim, E. H., Nies, D. H., McEvoy, M. M., & Rensing, C. (2011). Switch or funnel: how RND-type transport systems control periplasmic metal homeostasis. Journal of bacteriology, 193(10), 2381–2387. doi:10.1128/JB.01323-10 Available at: https://jb.asm.org/content/193/10/2381/figures-only?sid=7bfe37e6-f976-406e-a88d-c79a0dcc3d6f .
The above dsDNA substrate were desgined to have three components. There is a double terminator followed by a kanamycin resistance gene, and these two components were flanked by the homology sequence of the gene to be knocked out. When the recombination is successful, the substrate will be recombined to the homology site of the E. Coli genome and disrupt the target gene. Meanwhile, the inserted terminator can ensure the stop of transcription of the target gene and the kanamycin gene can act as a selection marker for sucessful recombination. (Fig. 3)
Fig. 3 The Lambda red recombineering process with our dsDNA substrate desgin.
These substrates can be recombined into plasmid vectors for storage. When in need, we can directly use PCR to produce the substrate in a large number.
Reference:
- Jafarian, V., & Ghaffari, F. (2017). A unique metallothionein-engineered in Escherichia coli for biosorption of lead, zinc, and cadmium; absorption or adsorption? Microbiology, 86(1), 73–81. doi: 10.1134/s0026261717010064
- Ueki, T., Sakamoto, Y., Yamaguchi, N., & Michibata, H. (2003). Bioaccumulation of Copper Ions by Escherichia coli Expressing Vanabin Genes from the Vanadium-Rich Ascidian Ascidia sydneiensis samea. Applied and Environmental Microbiology, 69(11), 6442–6446. doi: 10.1128/aem.69.11.6442-6446.2003
- Rensing, C., Fan, B., Sharma, R., Mitra, B., & Rosen, B. P. (2000). CopA: An Escherichia coli Cu(I)-translocating P-type ATPase. Proceedings of the National Academy of Sciences of the United States of America, 97(2), 652–656. doi:10.1073/pnas.97.2.652
- Padilla-Benavides, T., George Thompson, A. M., McEvoy, M. M., & Argüello, J. M. (2014). Mechanism of ATPase-mediated Cu+ export and delivery to periplasmic chaperones: the interaction of Escherichia coli CopA and CusF. The Journal of biological chemistry, 289(30), 20492–20501. doi:10.1074/jbc.M114.577668
- Franke, S., Grass, G., Rensing, C., & Nies, D. H. (2003). Molecular analysis of the copper-transporting efflux system CusCFBA of Escherichia coli. Journal of bacteriology, 185(13), 3804–3812. doi:10.1128/jb.185.13.3804-3812.2003
- Fong, S. , Camakaris, J. and Lee, B. T. (1995), Molecular genetics of a chromosomal locus involved in copper tolerance in Escherichia coli K‐12. Molecular Microbiology, 15: 1127-1137. doi:10.1111/j.1365-2958.1995.tb02286.x