Difference between revisions of "Part:BBa K1321005"

(→‎Usage and Biology)
(→‎Usage and Biology)
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===Usage and Biology===
 
===Usage and Biology===
 +
 +
{| class="wikitable" style="float:right; margin-left: 10px;"
 +
|+ align="bottom"|'''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].
 +
| align="center" style="background:#f0f0f0;"|'''Ion'''
 +
| align="center" style="background:#f0f0f0;"|'''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 [https://parts.igem.org/Part:BBa_K1321005#References (1)]. PCs are found mainly in plants but also in other organisms including diatoms, fungi, algae (including cyanobacteria) and some invertebrates ([https://parts.igem.org/Part:BBa_K1321005#References 1], [https://parts.igem.org/Part:BBa_K1321005#References 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 ([https://parts.igem.org/Part:BBa_K1321005#References 1], [https://parts.igem.org/Part:BBa_K1321005#References 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 [https://parts.igem.org/Part:BBa_K1321005#References (3)].  
 
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 [https://parts.igem.org/Part:BBa_K1321005#References (1)]. PCs are found mainly in plants but also in other organisms including diatoms, fungi, algae (including cyanobacteria) and some invertebrates ([https://parts.igem.org/Part:BBa_K1321005#References 1], [https://parts.igem.org/Part:BBa_K1321005#References 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 ([https://parts.igem.org/Part:BBa_K1321005#References 1], [https://parts.igem.org/Part:BBa_K1321005#References 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 [https://parts.igem.org/Part:BBa_K1321005#References (3)].  
Line 10: Line 30:
 
The main metal PCs confer tolerance to is Cadmium [https://parts.igem.org/Part:BBa_K1321005#References (1)] and EC20 peptide has showed good binding whilst fused to a cellulose binding domain (CBD) for water purification [https://parts.igem.org/Part:BBa_K1321005#References (4)] and when anchored to bacterial cell membrane to confer Cd2+ tolerance ([https://parts.igem.org/Part:BBa_K1321005#References 3], [https://parts.igem.org/Part:BBa_K1321005#References 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, [https://parts.igem.org/Part:BBa_K1321005#References 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 [https://parts.igem.org/Part:BBa_K1321005#References (7)], and to target CdSe/ZnS quantum dots to streptavidin expressing HeLa cells when biotinylated [https://parts.igem.org/Part:BBa_K1321005#References (8)].
 
The main metal PCs confer tolerance to is Cadmium [https://parts.igem.org/Part:BBa_K1321005#References (1)] and EC20 peptide has showed good binding whilst fused to a cellulose binding domain (CBD) for water purification [https://parts.igem.org/Part:BBa_K1321005#References (4)] and when anchored to bacterial cell membrane to confer Cd2+ tolerance ([https://parts.igem.org/Part:BBa_K1321005#References 3], [https://parts.igem.org/Part:BBa_K1321005#References 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, [https://parts.igem.org/Part:BBa_K1321005#References 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 [https://parts.igem.org/Part:BBa_K1321005#References (7)], and to target CdSe/ZnS quantum dots to streptavidin expressing HeLa cells when biotinylated [https://parts.igem.org/Part:BBa_K1321005#References (8)].
  
−
{| class="wikitable"
+
 
−
|+
+
We used this part to create a library of different cellulose-binding domains fused to phytochelatin:
−
|-
+
{| {{table}}
−
|'''Ion'''
+
| rowspan="2" align="center" style="background:#f0f0f0;"|'''Cellulose Binding Domain'''
−
|'''Approximate adsorption increase'''
+
| colspan="2" align="center" style="background:#f0f0f0;"|'''Promoter: LacI'''
 +
| colspan="2" align="center" style="background:#f0f0f0;"|'''Promoter: T7'''
 +
| colspan="2" align="center" style="background:#f0f0f0;"|'''Promoter: None'''
 
|-
 
|-
−
|Zn2+
+
|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)
−
|219 %
+
 
|-
 
|-
−
|Pb2+
+
| Linker-dCBD||[https://parts.igem.org/BBa_K1321110 K1321110]||-||[https://parts.igem.org/BBa_K1321100 K1321100]||-||[https://parts.igem.org/BBa_K1321090 K1321090]||-
−
|210 %
+
 
|-
 
|-
−
|Cu2+
+
| 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]
−
|76 %
+
 
|-
 
|-
−
|Cd2+
+
| 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]
−
|59 %
+
 
|-
 
|-
−
|Ni2+
+
| 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])
−
|45 %
+
 
|-
 
|-
−
|Mn2+
+
| CenA-Linker||-||[https://parts.igem.org/BBa_K1321117 K1321117]||-||[https://parts.igem.org/BBa_K1321107 K1321107]||-||[https://parts.igem.org/BBa_K1321097 K1321097]
−
|31 %
+
 
|-
 
|-
−
|Co2+
+
| None||[https://parts.igem.org/BBa_K1321007 K1321007]*||||[https://parts.igem.org/BBa_K1321006 K1321006]*||||[https://parts.igem.org/BBa_K1321005 K1321005]||
−
|marginal
+
|}
−
|}'''Table 1.''' Metal ion adsorption increase of ''C. metallidurans'' cells with PC EC20 displayed on cell surface, compared to negative control taken from a study by Biondo ''et al'' 2012 [6].
+
Bracketed: in progress - SDM to remove illegal EcoR1 site.
 +
"*" Still to ship to registry.
 +
"**" Part not cloned.
  
 
===Sequence and Features===
 
===Sequence and Features===

Revision as of 18:18, 14 October 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) and EC20 peptide has showed good 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:

Cellulose Binding Domain Promoter: LacI Promoter: T7 Promoter: None
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)
Linker-dCBD K1321110 - K1321100 - K1321090 -
CBDclos K1321111 K1321114 K1321101 K1321104 K1321091 K1321094
CBDcex K1321112 K1321115 (K1321102) K1321105 K1321092 K1321095
Linker-CBDcipA-Linker K1321113** (K132116) K1321103** (K1321106) (K1321093) (K1321096)
CenA-Linker - K1321117 - K1321107 - K1321097
None K1321007* K1321006* K1321005

Bracketed: in progress - SDM to remove illegal EcoR1 site. "*" Still to ship to registry. "**" Part not cloned.

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.