Difference between revisions of "Part:BBa K1321005"

 
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We used this part to create a library of different cellulose-binding domains fused to phytochelatin:
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We used this part to create a library of different cellulose-binding domains fused to phytochelatin ([http://2014.igem.org/Team:Imperial/Functionalisation project page]) as shown in the table below:
{| {{table}}
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[[File:IC14-PC-part-table.PNG]]
| rowspan="2" align="center" style="background:#f0f0f0;"|'''Cellulose Binding Domain'''
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| colspan="2" align="center" style="background:#f0f0f0;"|'''Promoter: LacI'''
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| colspan="2" align="center" style="background:#f0f0f0;"|'''Promoter: T7'''
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As part of our project, we needed to assay the metal binding capability of our Phytochelatins whilst fused to CBDs (cellulose binding domains). The parts used for this assay are
| colspan="2" align="center" style="background:#f0f0f0;"|'''Promoter: None'''
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Phytochelatin+CBDcex, Phytochelatin+dCBD, CBDcipA+Phytochelatin, Phytochelatin alone and sfGFP+dCBD wash (only one wash).
|-
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|N-terminal PC (PC-CBD)||C-terminal PC (CBD-PC)||N-terminal PC (PC-CBD)||C-terminal PC (CBD-PC)||N-terminal PC (PC-CBD)||C-terminal PC (CBD-PC)
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Phytochelatin fused to our CBDs were bound onto cellulose that were dried in the bottom of 96-well plates and tested against 3 different metals (nickel, copper, zinc).  
|-
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First, the fusion protein cell lysate was incubated overnight in the cellulose wells. Following this, the metal salt solutions are added in excess into the wells.  
| Linker-dCBD||[https://parts.igem.org/BBa_K1321110 K1321110]||-||[https://parts.igem.org/BBa_K1321100 K1321100]||-||[https://parts.igem.org/BBa_K1321090 K1321090]||-
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Finally, an EDTA step removes the bound metal ions into solution, and the metal concentration in solution is quantified by mass spectrometer. Multiple washes with PBS and water were done between each binding step, ensuring that the metal ions that are measured were released from the phytochelatin.
|-
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| CBDclos||[https://parts.igem.org/BBa_K1321111 K1321111]||[https://parts.igem.org/BBa_K1321114 K1321114]||[https://parts.igem.org/BBa_K1321101 K1321101]||[https://parts.igem.org/BBa_K1321104 K1321104]||[https://parts.igem.org/BBa_K1321091 K1321091]||[https://parts.igem.org/BBa_K1321094 K1321094]
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To evaluate if these CBD fusions were reusable, we re-applied metal ion solutions onto the same wells with the CBD fusions adhered. We washed the wells as before, then eluted with EDTA in the same manner. The results from the each elutions are shown below, with the final graph comparing between the first and second wash. We found along the way our bacterial cellulose alone had some metal chelating properties, as was also confirmed by our filtering set up with the dCBD-phytochelatin, but the second elution seems to show less background noise as the EDTA disrupts cellulose's natural binding to the metal ions. The full table of results are shown below as well. The full assay protocol can be found as ‘Metal binding assay protocol’ at this link:
|-
+
http://2014.igem.org/Team:Imperial/Protocols
| CBDcex||[https://parts.igem.org/BBa_K1321112 K1321112]||[https://parts.igem.org/BBa_K1321115 K1321115]||([https://parts.igem.org/BBa_K1321102 K1321102])||[https://parts.igem.org/BBa_K1321105 K1321105]||[https://parts.igem.org/BBa_K1321092 K1321092]||[https://parts.igem.org/BBa_K1321095 K1321095]
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|-
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| Linker-CBDcipA-Linker||[https://parts.igem.org/BBa_K1321113 K1321113]**||([https://parts.igem.org/BBa_K132116 K132116])||[https://parts.igem.org/BBa_K1321103 K1321103]**||([https://parts.igem.org/BBa_K1321106 K1321106])||([https://parts.igem.org/BBa_K1321093 K1321093])||([https://parts.igem.org/BBa_K1321096 K1321096])
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To read more about this assay and explanation of these results, please see:
|-
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http://2014.igem.org/Team:Imperial/Water_Filtration
| CenA-Linker||-||[https://parts.igem.org/BBa_K1321117 K1321117]||-||[https://parts.igem.org/BBa_K1321107 K1321107]||-||[https://parts.igem.org/BBa_K1321097 K1321097]
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|-
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[[File:IC14_first_wash_CBD_metal_conc.png|700px|left|]]
| None||[https://parts.igem.org/BBa_K1321007 K1321007]*||||[https://parts.igem.org/BBa_K1321006 K1321006]*||||[https://parts.igem.org/BBa_K1321005 K1321005]||
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|}
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[[File:IC14_second_wash_CBD_metal_conc.png|700px|left|]]
Bracketed: in progress - SDM to remove illegal EcoR1 site.
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"*" Still to ship to registry.
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[[File:IC14_Comparison_Nickel_conc_washes_CBDs.png|700px|left|]]
"**" Part not cloned.
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[[File:IC-2014_metalbindingtable.jpg|900px|left|]]
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===Sequence and Features===
 
===Sequence and Features===

Latest revision as of 04:33, 2 November 2014

Synthetic Phytochelatin EC(20) RFC[25]

Heavy metal-binding synthetic phytochelatin EC20 in Freiburg format (RFC[25]) to allow for easy use in fusion proteins.

Usage and Biology

Table 1. Metal ion adsorption increase of C. metallidurans cells with PC EC20 displayed on cell surface, compared to negative control. Taken from Biondo et al 2012 (6).
Ion Approximate adsorption increase
Zn2+ 219 %
Pb2+ 210 %
Cu2+ 76 %
Cd2+ 59 %
Ni2+ 45 %
Mn2+ 31 %
Co2+ marginal

Synthetic phytochelatins are analogues of phytochelatins (PCs); cysteine-rich peptides of the general structure (γ-GluCys)n-Gly (n~2-5) which bind and sequester heavy metals, with a role in detoxification (1). PCs are found mainly in plants but also in other organisms including diatoms, fungi, algae (including cyanobacteria) and some invertebrates (1, 2). Natively, the PCs are enzymatically synthesized by phytochelatin synthase (PCS) from a glutathione (GSH) precursor and the amino acids in the resulting peptide are linked by the non-standard gamma peptide bonds (1, 2). However, genetically encoded analogues (containing normal alpha-peptide bonds) of different number Glu-Cys (EC) repeats have been characterised, with the EC20 having the highest binding (3).

The main metal PCs confer tolerance to is Cadmium (1) with a reported stoichiometry of 10 Cd2+ per peptide (3). EC20 peptide has still showed good metal ion binding whilst fused to a cellulose binding domain (CBD) for water purification (4) and when anchored to bacterial cell membrane to confer Cd2+ tolerance (3, 5). PC EC20 has also been shown to bind many more heavy metals. For example, when displayed on the surface of Cupriavidus metallidurans, the cells capacity to adsorb ions increased by 31 to 219% depending on the ion (table 1, 6). These binding properties of PC EC20 have been utilised in applications including a regeneratable biosensor of Hg2+, Cd2+, Pb2+, Cu2+ and Zn2+ ions to 100 fM–10 mM (7), and to target CdSe/ZnS quantum dots to streptavidin expressing HeLa cells when biotinylated (8).


We used this part to create a library of different cellulose-binding domains fused to phytochelatin ([http://2014.igem.org/Team:Imperial/Functionalisation project page]) as shown in the table below: IC14-PC-part-table.PNG


As part of our project, we needed to assay the metal binding capability of our Phytochelatins whilst fused to CBDs (cellulose binding domains). The parts used for this assay are Phytochelatin+CBDcex, Phytochelatin+dCBD, CBDcipA+Phytochelatin, Phytochelatin alone and sfGFP+dCBD wash (only one wash).

Phytochelatin fused to our CBDs were bound onto cellulose that were dried in the bottom of 96-well plates and tested against 3 different metals (nickel, copper, zinc). First, the fusion protein cell lysate was incubated overnight in the cellulose wells. Following this, the metal salt solutions are added in excess into the wells. Finally, an EDTA step removes the bound metal ions into solution, and the metal concentration in solution is quantified by mass spectrometer. Multiple washes with PBS and water were done between each binding step, ensuring that the metal ions that are measured were released from the phytochelatin.

To evaluate if these CBD fusions were reusable, we re-applied metal ion solutions onto the same wells with the CBD fusions adhered. We washed the wells as before, then eluted with EDTA in the same manner. The results from the each elutions are shown below, with the final graph comparing between the first and second wash. We found along the way our bacterial cellulose alone had some metal chelating properties, as was also confirmed by our filtering set up with the dCBD-phytochelatin, but the second elution seems to show less background noise as the EDTA disrupts cellulose's natural binding to the metal ions. The full table of results are shown below as well. The full assay protocol can be found as ‘Metal binding assay protocol’ at this link: http://2014.igem.org/Team:Imperial/Protocols


To read more about this assay and explanation of these results, please see: http://2014.igem.org/Team:Imperial/Water_Filtration

IC14 first wash CBD metal conc.png
IC14 second wash CBD metal conc.png
IC14 Comparison Nickel conc washes CBDs.png
IC-2014 metalbindingtable.jpg














Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


References

1. Cobbett, C. & Goldsbrough, P. (2002) Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis. Annual review of plant biology. 53, 159–182.

2. Rea, P.A. (2012) Phytochelatin synthase: of a protease a peptide polymerase made. Physiologia plantarum. 145 (1), 154–164.

3.Bae, W., Chen, W., Mulchandani, A. & Mehra, R.K. (2000) Enhanced bioaccumulation of heavy metals by bacterial cells displaying synthetic phytochelatins. Biotechnology and bioengineering. 70 (5), 518–524.

4. Xu, Z., Bae, W., Mulchandani, A., Mehra, R.K., et al. (2002) Heavy Metal Removal by Novel CBD-EC20 Sorbents Immobilized on Cellulose. Biomacromolecules. [Online] 3 (3), 462–465.

5. Chaturvedi, R. & Archana, G. (2014) Cytosolic expression of synthetic phytochelatin and bacterial metallothionein genes in Deinococcus radiodurans R1 for enhanced tolerance and bioaccumulation of cadmium. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine. 27 (3), 471–482.

6. Biondo, R., da Silva, F.A., Vicente, E.J., Souza Sarkis, J.E., et al. (2012) Synthetic phytochelatin surface display in Cupriavidus metallidurans CH34 for enhanced metals bioremediation. Environmental science & technology. 46 (15), 8325–8332.

7. Bontidean, I., Ahlqvist, J., Mulchandani, A., Chen, W., et al. (2003) Novel synthetic phytochelatin-based capacitive biosensor for heavy metal ion detection. Biosensors and Bioelectronics. 18 (5-6), 547–553.

8.Pinaud, F., King, D., Moore, H.-P. & Weiss, S. (2004) Bioactivation and cell targeting of semiconductor CdSe/ZnS nanocrystals with phytochelatin-related peptides. Journal of the American Chemical Society. 126 (19), 6115–6123.