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Revision as of 12:56, 21 October 2009

Transport

To isolate heavy metals from the environment we require uptake systems. So far we found several different mechanisms to create such a system. We investigated three kinds of metal uptake:

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

We have investigated several systems to determine which are suitable for the final design. The following systems are under consideration:

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

We chose to focus on GlpF and HmtA, the final device was made with GlpF for arsenate purification.

Arsenite uptake via GlpF

GlpF

Introduction

GlpF is an aquaglycerol porin of E.coli which facilitates not only glycerol import, but also arsenic (As) and antimone (Sb) import (Fu, DX, et al.2000), (Meng, YL, et al.2004), (Porquet, A, et al.2007), (Rosen, BR, et al.2009) . It has homologues in other organisms; Fps1p has shown to facilitate arsenic import in yeast and AQP9 is the mammalian homologue (Porquet, A, et al.2007), (Rosen, BR, et al.2009) . The GlpF aquaglycerol porin is a membrane protein with a symmetric arrangement of four independent GlpF channels. One monomer of this tetramer GlpF porin consists of six transmembrane and two half membrane-spanning α-helices that form a right-handed helical bundle around the channel. The channel has a diameter of ~15Å at the periplasmid end, which constricts towards a diameter of ~3.8Å at the beginning of a 28 Å long selective channel that ends at the cytoplasmic end (Fu, DX, et al.2000). The GlpF is a stereospecific channel that is thought to be more selective on molecular size than on chemical structure (Fu, DX, et al.2000, (Heller, KB, et al.1980) . It does allow transport of a variance of non-charged compounds ranging from polyhydric alcohols, glycerol being one of them, arsenic to antimone (Fu, DX, et al.2000), (Meng, YL, et al.2004), (Porquet, A, et al.2007), (Rosen, BR, et al.2009), (Heller, KB, et al.1980). Carbon sugars and ions are shown to be unable to be transported by GlpF (Heller, KB, et al.1980). At physiological pH arsenic and antimone are not present in their ionic state but rather as As(OH)3 and Sb(OH)3 (Rosen, BR, et al.2009). These elements show a charge distribution similar to glycerol and a smaller but comparable volume. The structural similarities are thought to be the reason for the possibility of these elements to enter the cell by GlpF (Porquet, A, et al.2007), GlpF facilitates transport of these compounds down there gradient (inside or outside the cell). If GlpF behaves as a nonsaturable transporter, a transport rate of 1umol of glycerol is transported per minute per mgr of cell protein (Heller, KB, et al.1980). The transport rate of GlpF for arsenic is estimated to be……

Cloning strategy

This part has been obtained from the genome of E.coli 356 in two steps with PCR. First the whole gene was obtained from the genome by using PCR and in the second step an EcoR1 restiction site was removed. The GlpF PCR product was restricted with XbaI and PstI and a psB1AC3 vector with a pMed promotor was restricted with SpeI and PstI. The restriction products were ligated. This resulted in a psB1AC3 vector with a promotor and GlpF.

Results

The ability of GlpF (overexpressed under lactose induction) to transport As(III) was tested by an arsenite uptake assay. Also the full accumulation device () was tested using this assay. Data and analysis can be found here. The expression of GlpF was not tested by SDS-PAGE or protein purification.

Modelling uptake GlpF

The import of As(III) via GlpF is modelled as a simple import reaction with Michaelis-Menten kinetics, in part because this makes it easy to specify, but also because we only have very high level data. The following allows a comparison with the data acquired from figure 1B from Meng 2004.

To determine the constants v5 and K5 we performed the following steps:

1. Read the wild-type line in figure 1B of Meng 2004 by pasting it in a drawing program and aligning/scaling the axes and then manually determining the coordinates of each data point.
2. Converted to units of concentration using the data in Meng 2004 and [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi the CCDB] (assuming that the cells are resting/non-growing), see our [http://spreadsheets.google.com/pub?key=t4gilzCbEaCFAvpEVWUE_zQ Google Docs spreadsheet]. Here we disregarded the fact that the measurements were made by taking out 0.1mL samples, as this does not change the concentrations. Specifically (note that uptake is in nmol/mg):
   • uptake_total (nmol) = uptake · 8mg · 0.3
     The ratio between dry and wet weight is 0.3 (see the [http://gchelpdesk.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB]).

      i 
   • As(III)_ex (µM=nmol/mL) = (10nmol/mL · 1mL - uptake_total) / (1.1-0.0073)mL
     The experiment started with 1mL of a 10µM=10nmol/mL solution of As(III). After adding the cells the total volume of the solution was 1.1mL, and 0.0073mL is an estimate of the total volume of cells in the solution, see below.

      i 
3. Fit the Michaelis-Menten equation to find the constants v5 and K5 in Mathematica (see [http://igemgroningen.googlecode.com/svn/trunk/buoyant/Models/Meng2004%20Figure%201B.nb the Mathematica notebook in SVN]) using the method from Goudar 1999 (a least squares fit of a closed-form solution of the differential equation).

Missing information/To Do

• Expression assesment
  • Stability
  • Level
• Functional assesment
  • Uptake speed
  • Affinity
  • Electrolyte potential generating force
• Q:Eliminate BioBrick restriction sites
• Q: What does the ars operon of our E. coli look like? Do we have both ArsA and ArsB? (And what about ArsR and ArsD?) A: We only have ArsRBC, see our BLAST results.

Additional sources

• Meng 2004 (As(III) and Sb(III) Uptake by GlpF and Efflux by ArsB in Escherichia coli)
• Rosen 2009 (Transport pathways for arsenic and selenium: A minireview)
• Porquet, A, et al.2007 (structural similarity between As(OH)3 and glycerol)
• Fu, DX, et al.2000 (Structure of the GlpF channel)
• Heller, KB, et al.1980 (Glycerol transport properties of GlpF)

Copper/zinc uptake via HmtA

HmtA

HmtA(heavy metal transporter A) from Pseudomonas aeruginosa [http://www.ncbi.nlm.nih.gov/protein/81857196 Q9I147] is a P-type ATPase importer. This membrane protein mediates the uptake of copper (Cu) and zinc (Zn) and was functionally expressed in E.coli ([http://www.ncbi.nlm.nih.gov/pubmed/19264958 Lewinson 2009]). We want to use this membrane protein to accumulate copper and zinc into the cells. we believe this atp driven transport is effectivent enough to sequester the medium from these compounds.

Cloning strategy

There are several restriction sites to be modified from [http://www.ncbi.nlm.nih.gov/pubmed/19264958 Lewinson's] pBAD construct. A vector with amp resistance with L-arabinose inducible HmtA-6HIS. The restriction sites can be modified without changing the amino acid sequence of the protein. We will create these mutations via PCR than digest the old methylated template and clone the product into competent cells. Create ON cultures for plasmid isolation for restriction check and repeat the process.

First we clone the gene into an iGEM vector pSB1AC3 and we lose the HIS tag and introducing one mutation to eliminate one EcoRI site. Than via PCR eliminate the PstI restriction site with proper primers and destroy the template with DnpI and transform into cells, repeat for the other PstI site.

Results

First results!

So far we have cloned HmtA as a biobrick without EcoRI site in the coding region into the iGEM vector. Unfortunately a mutation occurred at base 103 from the start of the orf. By a point mutation c to t in the first nucleotide of the codon changed arginine 35 to a Cysteine. We do not know the effects but we suspect it might have a great influence due to the very reactive sidechain, eventhough it is not in the channel itself based on [http://www.cbs.dtu.dk/services/TMHMM/] TMHMM] predictions witch indicate trans membrane segments of a protein. Further cloning is expected to unsuccessful because the iPTG induced clones grow even better than Controle most likely because of missing pLAC-RBS. There was no positive controle with the L-arabinose inducable HmtA-6His in pBAD. We did do expression experiments with the pBAD construct to purified the membrane protein as quality controle.

Missing information/To Do

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

Heavy metal uptake coupled to citrate via efCitH bsCitM

Citrate uptake coupled to heavy metals enables forcefeeding of the toxic compounds into the cell when citrate is the only carbon source available. This could be a very efficient strategy to accumulate vast 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

Additional sources

More information on this topic can be found in:

Bastiaan Krom. Citrate transporters of Bacilus subtilis PhD thesis. http://dissertations.ub.rug.nl/faculties/science/2002/b.p.krom/ Dissertation Groningen

Jessica B. Warner. Regulation and expression of the metal citrate transporter CitM PhD thesis. http://dissertations.ub.rug.nl/faculties/science/2002/j.b.warner/ Dissertation Groningen

Periplasmic accumulation of heavy metals via Mer Operon

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 (i.e. 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 (Pennella 2005, Kao 2008).

Missing information/To Do

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

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 (both for the "normal" cell and the cell with overexpression of the transporter)
    • 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.
    • How fast does it transport metal in/out the cell.
      • Set up tests with (initial) extracellular concentrations of about ¹/₃K (25% of V_max), K (50% of V_max), 3K (75% of V_max) and 10mM (99.7% of V_max, corresponding to extremely polluted water), and a control with no arsenic. Obviously, more tests is better. In general a desired fraction of V_max at the initial concentration can be attained by using an initial concentration of x/(1-x) times K.
      • Determine "final" (steady-state) concentration of As(III) in the solution and in the cells. (Concentration over time is even better!)
      • This means that the total volume of the cells (and the solution) has to be determined. Possibly through looking at the dry weight (without arsenic!).
      • By manipulating the equation for the derivative of As(III) in equilibrium, As(III) can be expressed as a function of As(III)_ex (given the V and K constants). We can try to fill in the computed V and K constants for GlpF and then use a least squares fit to estimate the V and K constants for ArsB.
      • NOTE: Interestingly Kostal 2004 already did an experiment like this with cells that overexpressed ArsR. We're looking at analysing these results under the assumption that overexpressing ArsR only gives a constant factor more accumulation (for 1-100&microM As(III)), but it would be very nice to do this ourselves for unmodified cells to determine whether this is indeed true (and to determine the factor).

Export of arsenicum via Ars operon

GlpF is the importer of arsenicum. After arsenicum enters the cell, in response the Ars operon produces ArsR. At the same time, ArsB is also produced by Ars operon. This happens because the Ars operon contains three open reading frames: the first is ArsR, second ArsB and the last one is ArsC. ArsB is the exporter of arsenicum. The ars operon is located on the chromosomal DNA of E. coli. For more information see: [http://biocyc.org/ECOLI/NEW-IMAGE?type=GENE-IN-CHROM-BROWSER&object=EG12235 biocyc].