Team:Groningen/Project/Transport

From 2009.igem.org

(Difference between revisions)
(Undo revision 6207 by Wilfred (Talk))
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*Copper /zinc uptake via HmtA
*Copper /zinc uptake via HmtA
-
*heavy metal uptake coupled to citrate via ''ef''CitH
+
*heavy metal uptake coupled to citrate via ''ef''CitH ''bs''CitM
*Arsenite uptake via GlpF
*Arsenite uptake via GlpF
*Periplasmic accumulation of heavy metals via Mer Operon.
*Periplasmic accumulation of heavy metals via Mer Operon.
Line 58: Line 58:
Lewinson O., Lee A.T., Rees D.C. 2009. A P-type ATPase importer that discriminates between essential and toxic transition metals. PNAS. vol. 106, no. 12, p. 4677-4682.
Lewinson O., Lee A.T., Rees D.C. 2009. A P-type ATPase importer that discriminates between essential and toxic transition metals. PNAS. vol. 106, no. 12, p. 4677-4682.
-
==Mer operon==
+
==Citrate coupled uptake==
 +
Citrate uptake coupled to heavy metals enables forcefeeding of the toxic compounds into the cell when citrate is the only carbound source available. This could be a very effecient strategy to accumelate vass ammounts of heavy metals.
 +
The current candidates are CitM from ''Bacilus subtilis'' and CitH form ''Enterococcus faecalis''
 +
 +
===Missing information/To Do===
 +
*Expression assesment
 +
**Stability
 +
**Level
 +
*Functional assesment
 +
**Uptake speed
 +
**Affinity
 +
**Electrolyte potential generating force
 +
*Eliminate BioBrick restriction sites
-
===Missing information===
 
===Literature===
===Literature===
-
 
+
Blancato, V.S., Magni, C. & Lolkema, J.S. Functional characterization and Me<sup>2+</sup> ion specificity of a Ca<sup>2+</sup>&#x2013;citrate transporter from <i>Enterococcus faecalis</i>. FEBS Journal 273, 5121-5130(2006).
-
Lewinson O., Lee A.T., Rees D.C. 2009. A P-type ATPase importer that discriminates between essential and toxic transition metals. PNAS. vol. 106, no. 12, p. 4677-4682.
+
Bastiaan krom Citrate transporters of Bacilus subtilis Proefschrift.
-
 
+
Jessica B. Warner Regulation and expression of the metal citrate transporter CitM Proefschrift.
==GlpF==
==GlpF==
-
===Missing information===
+
===Missing information/To Do===
 +
*Expression assesment
 +
**Stability
 +
**Level
 +
*Functional assesment
 +
**Uptake speed
 +
**Affinity
 +
**Electrolyte potential generating force
 +
*Eliminate BioBrick restriction sites
 +
 
===Literature===
===Literature===
1. Meng, Y., Liu, Z. & Rosen, B.P. As(III) and Sb(III) Uptake by GlpF and Efflux by ArsB in Escherichia coli. J. Biol. Chem. 279, 18334-18341(2004).
1. Meng, Y., Liu, Z. & Rosen, B.P. As(III) and Sb(III) Uptake by GlpF and Efflux by ArsB in Escherichia coli. J. Biol. Chem. 279, 18334-18341(2004).

Revision as of 14:32, 7 June 2009

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Introduction

We are trying to find suitable systems capable of isolating heavy metals from the environment. There are several different mechanisms to achieve such a thing. We examined 3 kinds:

  • Metal transporters, that transport the metal from the environment (ie. wastewater) to the cytoplasm
    • Uncoupled
    • Coupled with 'helper' protein
  • Metal binding proteins in the periplasm

We will investigate severals systems, to find which are suitable for the final design. the following systems are under concideration.

  • Copper /zinc uptake via HmtA
  • heavy metal uptake coupled to citrate via efCitH bsCitM
  • Arsenite uptake via GlpF
  • Periplasmic accumulation of heavy metals via Mer Operon.

HmtA

Time-dependent copper uptake. Cells transformed with empty plasmid (squares) or HmtA-encoding plasmid (all other traces) were cultured in the absence of metals, washed with metal-free buffer, and allowed to recover in the presence of glucose. Transport was initiated by the addition of 250 nM CuCl2, and samples were withdrawn at the indicated times. Where indicated, 0.5 mM DTT or 0.25 mM cysteine was included in the reaction mixture. Total internal metal concentrations were measured by inductively coupled plasma mass spectroscopy. Error bars represent standard deviations of three repeats.

HmtA, heavy metal transporter A from Pseudomonas aeruginosa Q9I147 is a P-type ATPase importer. It mediates the uptake of Copper (Cu) and Zinc (Zn) and is functionally expressed in E.coli.

>gi|81857196|sp|Q9I147|Q9I147_PSEAE Probable cation-transporting P-type ATPase

Enzyme Number of Sites
EcoRI 0
XbaI 0
NotI 0
SpeI 0
PstI 2


Missing information/To do

  • Expression assesment
    • Stability
    • Level
  • Functional assesment
    • Uptake speed
    • Affinity
    • Electrolyte potential generating force
  • Eliminate BioBrick restriction sites

Literature

Lewinson O., Lee A.T., Rees D.C. 2009. A P-type ATPase importer that discriminates between essential and toxic transition metals. PNAS. vol. 106, no. 12, p. 4677-4682.

Citrate coupled uptake

Citrate uptake coupled to heavy metals enables forcefeeding of the toxic compounds into the cell when citrate is the only carbound source available. This could be a very effecient strategy to accumelate vass ammounts of heavy metals. The current candidates are CitM from Bacilus subtilis and CitH form Enterococcus faecalis

Missing information/To Do

  • Expression assesment
    • Stability
    • Level
  • Functional assesment
    • Uptake speed
    • Affinity
    • Electrolyte potential generating force
  • Eliminate BioBrick restriction sites


Literature

Blancato, V.S., Magni, C. & Lolkema, J.S. Functional characterization and Me2+ ion specificity of a Ca2+–citrate transporter from Enterococcus faecalis. FEBS Journal 273, 5121-5130(2006). Bastiaan krom Citrate transporters of Bacilus subtilis Proefschrift. Jessica B. Warner Regulation and expression of the metal citrate transporter CitM Proefschrift.

GlpF

Missing information/To Do

  • Expression assesment
    • Stability
    • Level
  • Functional assesment
    • Uptake speed
    • Affinity
    • Electrolyte potential generating force
  • Eliminate BioBrick restriction sites


Literature

1. Meng, Y., Liu, Z. & Rosen, B.P. As(III) and Sb(III) Uptake by GlpF and Efflux by ArsB in Escherichia coli. J. Biol. Chem. 279, 18334-18341(2004).
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