Team:Groningen/Project/Accumulation

From 2009.igem.org

Introduction

Once heavy metals have entered the cell, it is crucial to keep them there. As these metals are toxic to cell survival in critical amounts, evolution has provided us with biological detoxicification proteins such as [http://en.wikipedia.org/wiki/Metallothionein metallothioneins]. These proteins can aid us in our quest to accumulate a variaty of heavy metals as they bind to a wide range of metals including cadmium, zinc, mercury, copper, arsenic, silver, etc..

Metallothioneins

Metallothioneins are a class of low molecular-weight metal-binding proteins (<10kDa) rich in cysteines residues(~30%). They are capable of binding a variety of heavy metals (e.g. Zn, Cu, Cd, Hg, As) with high avidity(Kb) (Merrifield2004) and they have readily been used to create cell based systems for purification of contaminated water Chen 1998, Brady 1994. In addition to their wide application possibilities, they also have the capacity to carry multiple metal ions at one time, in contrast to some other metalloproteins that carry them one-on-one Chang 1998. Many forms of metallothioneins are known and their affinity for different metals has been investigated on several occasions, such as for cadmium Deng 2007, arsenic Ngu 2006, Kostal 2004, Singh 2008, mercury Chen 1998, Chen 1997-2, Deng 2008, nickel Deng 2003 or a combination of metals Chang 1998, Kao 2008. Metal-protein complexes can be quantified using a fluorescent molecule Cadosch 2008.

Cloning strategy

Fig. 1: Cloning strategy for the metal accumulation device. A promoter taken from J61002 will be cloned in front of a metallothionein and a metal transporter in a pSB1AC3 vector. This device will be combined with the floating device.

In order to have a functional accumulation device, a metallothionein (MT) will be produced by PCR and cloned into pSB1AC3, also a corresponding metal-ion transporter was produced by PCR and cloned behind the MT. Both will expressed by one promoter (constitutive or lactose inducible). In this way the bacterium will take up the metal-ion and consecutively the metal-ion will be sequestered by the MT. When this device is combined with the floating device, the bacteria will start floating when a certain treshold of intracellular metal concentration is reached, because the negative regulator of the bouyancy device will be released and the gas vesicle cluster can be transcribed.

Metals

Arsenic For the accumulation of arsenic some MTs are possible, like rh-MT (human MT) Ngu 2006 and fMT (the seaweed species fucus vesiculosis) both binding As(III). The oxidized version of arsenic (As (V)) can also be bound by the metallothioneins but with lower affinity Singh 2008, another way As(V) is proposed to be accumulated is by conversion of As(V) to As(III) by the arsenate reductase and subsequent bound to the metallothionein or ArsR. rh-MT is known to bind 6x As(III) per molecule, fMT binds 5x As(III). No extra quantitative information is known from literature. ArsR is a trans-acting repressor that senses environmetal As(III)and regulates the chromosomal Ars operon. The ArsR protein has a specific binding site for As(III) and descriminates effectively against other metals like: phosphate, cadmium, sulfate and cobalt. The affinity of ArsR for As(III) is very high 10^-15M of AS(III) can induce the promotor. The specific binding site spans 33 nucleotides in the promotor region including the putative -35 promotor element. When ArsR was purified, it's size corresponded to that of a homodimer, bound to promoter DNA. Because of the high affinity of ArsR for As(III) the protein could be used for arsenic remediation. Chen and co-workers overexpressed ArsR in E.Coli JM109 cells and found that the specific AS(III) content was 13-fold higher than the control without ArsR expression. High level expression of ArsR appeared to be toxic as a 3-fold reduction in cell density was observed. It has been shown that fusion partners reduce the toxicity of overexpression. Originally, Chen and co-workers made a fusion between ArsR and ELP(elastin protein), which is build out of VPGVG repeats. Because making a ArsR ELP153 fusion is very time consuming, we choose to make a fusion between MBP (maltose binding protein) and ArsR Chen 1998.

Also see the sensitive promoters. As ordering rh-MT was not succesfull, we try to use fMT for accumulation of As(III) and use ArsR to regulate the expression of the GVP cluster behind the ArsR regulated promoter.

Copper

pBG68 with mymT (M. tuberculosis MT gene for Cu(I) accumulation) Gold 2008

• • Vector properties: pMB1 ori(20 copy nr.), M13 ori (? copy nr.), tagged with mxe-gyrA intein and chitin binding domain, produced from IPTG inducible T7 promoter (LacI also present).

Zinc

• Zn--> pMHNR1.1 (a pET29a vector) with smtA (Cyanobacterial MT gene for Zn accumulation) Blindauer2001

Alternatives Inclusion bodies Fowler 1987
(Bacterio)Ferritins
Phytochelatins
[http://www.wiley.com/legacy/products/subject/reference/messerschmidt_toc.html A list of opportunities]

Inhibitory characteristics?

Modelling

Arsenic - ArsR

Below you can calculate how many grams of arsenic will be taken out of the water per cubic meter of cells. This extra weight raises the density of the cell and therefore lowers its capacity for buoyancy. Our preliminary results look very promising. Even under the assumption that the weight of the metal is added to the weight of the cells, without increasing their volume, we could add upto a hundred times the currently computed weight without having a large effect on the required fraction of gas vesicles (it will only go up from about 12.2% to 12.7%).

At this moment we use four different variables:

1. Molecular weight of arsenic. Source: [http://en.wikipedia.org/wiki/Arsenic Arsenic page on Wikipedia]
2. Millimol arsenic per kg of cell dryweight (note that this is equivalent to nmol/mg). Source: Kostal2004
3. The proportion between the weight of a dry cell and a wet cell. Source: [http://redpoll.pharmacy.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi CCDB Database]
4. Cell density. Source: see our gas vesicle page.

As per cell volume = awAs * nAs(III) /
   Mcell(dry) * Mcelldrywet * rhocell
mol As per cell volume = nAs(III) / 
   Mcell(dry) * Mcelldrywet * rhocell

At a lower level arsenic accumulation can be described using reactions between ArsR, As(III) and the ars promoter. As shown in the figure on the right a number of different substances(/complexes) are involved. For our purposes it is especially important to determine what fraction of As(III) is unbound, if more As(III) is bound we can accumulate more.

In addition to binding to As(III), ArsR can repress ars, creating a negative feedback loop. In effect this regulates the production of ArsR based on the As(III) concentration Chen1997. In the E. coli top10 there is only ars promoter present on the genome to produce ArsR (see BLAST results). There are plasmids which produce both ArsR and ArsD, but these are not used in this project. We intend to introduce instead a constitutive promoter (pro), which produces just ArsR, in order to bind as much As(III) as possible.

The calculator below tries to compute the ratio between bound and unbound arsenic, specifically As(III), in the cell. See our Modelling page for detailed information on the constants/variables used and a derivation of the formulas. Note that the computations currently involve slightly more variables/constants than strictly necessary. TODO: The half-lifes were guesses based on cell-division, but since we have "resting" cells which we assume do not divide this seems like a very bad guess, so we need a new guess?

In conclusion:

• Even at the accumulation levels of Koster et al. the amount of arsenic accumulated in E. coli is so little that it shouldn't matter much for the buoyant density (which normally is about 1100kg/m³).
• If you substitute constitutive promotors for ars promotors you can see that it is clearly advantageous to use constitutive promotors as just adding ars promoters does not increase the accumulation factor. A with a plasmid containing an ars promoter and (just) a gene coding for ArsR behind it might contain more arsenic, but there would also be more unbound arsenic, increasing the toxicity.
• The model is not very sensitive to different values for K3d (with K3d=1mM the accumulation factor is 248.06 and with K3d=1nM it is 240.57).
• The accumulation factor is greatly affected by the product of the half-life of ArsR and the production rate.

Planning and requirements:

• Modelling
  • Speed
  • Metaliotheines concentration
  • How often does the ArsR sensitive operator/operon occur in our E. coli?
• Lab
  • Measurements
    • Transport Assays
      • Protein expression levels determined by immunoblotting using anti-ArsA and anti-ArsD antibodies Lin2007
      • Inductively coupled mass spectrometry (ICP-MS) Meng2004
    • Measure accumulation. By measuring before/after concentration metal with and without accumulation protein.
    • Determine the dissociation constant of ArsR and As(III). (By measuring the ratio between bound and unbound ArsR?)
      • It might be possible to do this with (tryptophan related) fluorescence (that is how it is done for ArsD in Chen1997). In the paper ArsD is purified, but if that's not feasible for us we might try to simply do it in living cells (and hope that ArsR both fluoresces enough and is produced enough to be measurable).
    • Production rate of ArsR?
  • Biobrick Bba_K129004
  • Rest