Team:Groningen/Project/Transport

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  <h1>Transport</h1>
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'''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:'''
'''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:'''
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*Metal transporters, Membrane proteins that transport the metal from the environment (<i>i.e.</i> wastewater) to the cytoplasm
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*Metal transporters, Membrane proteins that transport the metal from the environment (<i>e.g.</i> wastewater) to the cytoplasm
**Uncoupled
**Uncoupled
**Coupled with 'helper' compounds
**Coupled with 'helper' compounds

Revision as of 20:20, 21 October 2009

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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 (e.g. 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).

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. RestictioLigationGlpF.JPG

Results

The ability of GlpF (overexpressed under IPTG induction) to transport As(III) was tested by an arsenite uptake assay. Also the full accumulation device (BBa_K190038) was tested using this assay. Data and analysis can be found here.

Growth WT.gif
Growth GlpF.gif
Growth GlpF fMT.gif

The graphs above represent the result of the membrane protein assay. The lines in the graphs represent the average optical density of a construct over time. The graph on the left show that increased As(III) levels inhibit growth and, that as more As(III) is added the lower the plateau is.

The middle graph is from the pLac GlpF construct. The curves are less steep in the log phase compared to WT because of the protein expresion by IPTG induction. In the absence of As(III) the plateau level equals the WT. If arsenite is present the plateaus are lower (OD600 <0.8) compared to WT. This is due to As(III) uptake by GlpF.

In the graph on the right we see the curves of low constitutively expressed GlpF and fMT and it shows a similar slope in the log phase compared to pLac GlpF due to protein expression and like WT 0 μM As(III) it has its plateau over OD600 0.9. If arsenite is present the plateaus are lower (OD600 <0.8) compared to WT. This is due to As(III) uptake by GlpF. Here the reduced growth is also an indicator for arsenite uptake. It is difficult to see if fMT has an effect because this assay can not show where the arsenite is and how fMT interferes with the cells detoxificatoin.

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.

Initial values
As(III)ex = µM
                (10µM · 1mL / 1.092mL)
Volumes
Vtotal = mL
Vcells = mL
                (0.1ml · 80mg/mL / 1100mg/mL)
E. coli has a density of approximately 1100mg/mL, see our gas vesicle page for more information.
 i 
Kinetic Constants
v5 = µmol/(s·L)
K5 = µM

Loading graph...

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 the CCDB (assuming that the cells are resting/non-growing), see our 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):
    • uptaketotal (nmol) = uptake · 8mg · 0.3
      The ratio between dry and wet weight is 0.3 (see the CCDB).
       i 
    • As(III)ex (µM=nmol/mL) = (10nmol/mL · 1mL - uptaketotal) / (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 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


Copper/zinc uptake via HmtA

HmtA

Introduction

HmtA(heavy metal transporter A) from Pseudomonas aeruginosa 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 (Lewinson 2009). We want to use this membrane protein to accumulate copper and zinc into the cells. we believe this ATP-driven pump is capable of generating an elevated intracellular concentration of these compounds enabling the harvesting of copper and zinc from the medium.

Cloning strategy

There are several restriction sites to be modified from Lewinson's pBAD construct. A vector with amp resistance with L-arabinose inducible HmtA-6HIS. The restriction sites have been silently mutated maintaining the amino acid sequence. We will create these mutations via PCR than digest the old methylated template and clone the product into competent cells.

Results

HmtA-6HIS on SDS-page

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 side chain of Cysteine, eventhough it is not in the channel itself based on TMHMM predictions which indicate trans membrane helices of a protein. Further cloning is expected to be unsuccessful because the iPTG induced clones grow even slightly better than the empty vector control. This is most likely cause by the missing pLAC-RBS in front of the gene. 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. result shown in the figure on the right.

Heavy metal uptake coupled to citrate via efCitH bsCitM

Force feeding of the heavy metals into the cell is possible when citrate is the only available carbon source. Citrate in complex with heavy metals can be translocated over the membrane into the cell via citrate transporters. This can be a very efficient strategy to accumulate vast ammounts of heavy metals. The two membrane proteins are CitM from Bacillus subtilis studied by B.P Krom. BsCitM can transport citrate in complex with Mg2+, Ni2+, Mn2+, Co2+, and Zn2+. The other is CitH from Enterococcus faecalis described by V.S Blancato. EfCitH catalyzes translocation of the citrate in complex with Ca2+, Sr2+ Mn2+ Mn2+ Cd2+ and Pb2+.


Additional sources

More information on this topic can be found in:

Bastiaan Krom. Citrate transporters of Bacillus subtilis PhD thesis. [Dissertation Groningen]

Jessica B. Warner. Regulation and expression of the metal citrate transporter CitM PhD thesis. [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 Vmax), K (50% of Vmax), 3K (75% of Vmax) and 10mM (99.7% of Vmax, corresponding to extremely polluted water), and a control with no arsenic. Obviously, more tests is better. In general a desired fraction of Vmax 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: biocyc.

ArsRBC operon.PNG