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 Transport was initiated by the addition of 250 nM CuCl2, and samples were withdrawn at the indicated times.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

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

73As(III) and 125Sb(III) uptake into cells of E. coli is facilitated by the aquaglyceroporin channel 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).
2. Rosen2009 (Transport pathways for arsenic and selenium: A minireview)

MerR family

Periplasmic accumulation of heavy metals via Mer proteins enables the harvesting of heavy metals from the medium. By binding the cytosolic and periplasmic metals to metallothionein and transporting the metal-protein complex into the periplasm. The MerR family consists of different proteins for one specific metal. (ie. PbrR(Lead), CueR(Copper), ZntR(Zinc), MerR(Mercury), ArsR(Arsenic), CadR(Cadmium))

As the cells die after uptake of Mg (and induction of the Mer transporter) this system is not very well usable for our project. The dead cells will not produce the gas vesicles (it may be used however by having the gas vesicles consitutively expressed), thereby bouyancy may be a problem.

Missing information/To Do

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

Literature

Kao, W., Huang, C. & Chang, J. Biosorption of nickel, chromium and zinc by MerP-expressing recombinant Escherichia coli. J. Hazard. Mater 158, 100-106(2008).

Pennella, M.A. & Giedroc, D.P. Structural determinants of metal selectivity in prokaryotic metal-responsive transcriptional regulators. Biometals 18, 413-428(2005).

Planning and requirements:

• Modelling:
  • Import speed
  • Amount
  • Max
• Lab:
  • HmtA
    • Zn/Cu alone
    • B-type ATPase (could be use if there is a ATP shortage?)
  • CitM (probably not used)
    • Divalent ions
    • Citrate around
    • Citrate can bind metals that are already bound.
  • Measurements
    • Transporter, on/off mechanism, up to what concentration (in the cell) does it still have metal uptake.
    • Measure concentration of metal. difference between begin and end concentrations of metal outside the cell