Team:Groningen/Project/Transport

From 2009.igem.org

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
• 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 Transport was initiated by the addition of 250 nM CuCl2, and samples were withdrawn at the indicated times.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

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.

Mer operon

Missing information

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.

GlpF

Missing information

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).

Heavy metal accumulation