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Team:Groningen/Project/Accumulation
From 2009.igem.org
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- Transport
- Accumulation
- Metal-sensitive Promoters
- Gas Vesicles
Introduction
Once heavy metals have entered the cell it is key 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 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 rich in cysteines residues. They are capable of binding a variety of heavy metals. And they have readily been used to create cell based systems for purification of contaminated water [2][3]. 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[4]. Many forms of metallothioneins are known and their affinity for different metals has been investigated on several occasions, such as for cadmium[5], arsenic [6][7][8], mercury[1][9][10], nickel[11] or a combination of metals[3][12]. Metal-protein complexes can be quantified using a fluorescent molecule[13].
Alternatives
Inclusion bodies[14]
(Bacterio)Ferritins
Phytochelatins
A list of opportunities
Inhibitory characteristics?
Modelling
Arsenic
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 buoyancy capacity. 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:
- Molecular weight of arsenic. Source: Arsenic page on Wikipedia
- Millimol arsenic per kg of cell dryweight (note that this is equivalent to nmol/mg). Source: Koster et al
- The proportion between the weight of a dry cell and a wet cell. Source: CCDB Database
- Cell density. Source: see our gas vesicle page.
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awAs(III) = g/mol Mcell(dry) / Mcell(wet) = ρcell = kg/m3 |
As(III) intake per volume of cells = g/m3 = µmol/liter ({{todo}} check) |
The following tries to compute the relation between bound and unbound arsenic, specifically As(III), in the cell. See our Modelling page for information on the constants/variables used.
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K1d = µM (???) K2d = µM K3d = µM K4d = µM τ1 = min (???) τ2 = min (???) |
ArsR = µM ArsD = µM ArsRAs = µM ArsDAs = µM ArsRop = µM ArsDop = µM |
Planning and requirements:
- Modelling
- Speed
- Metaliotheines concentration
- Lab
- Measurements
- Measure accumulation. By measuring before/after concentration metal with and without accumulation protein.
- Biobrick Bba_K129004
- Rest
- Measurements
Literature
- Brady et al.:The use of hollow fiber cross-flow microfiltration in bioaccumulation and continuous removal of heavy metals from solution by Saccharomyces cerevisiae, Biotechnology and bioengineering (1994) 44(11);1362-1366
- Cadosch et al.: Uptake and intracellular distribution of various metal ions in human monocyte-derived dendritic cells detected by Newport Green DCF diacetate ester Journal of Neuroscience Methods (2009) 178(1);182-187
- Chang et al.:Cysteine contributions to metal binding preference for Zn/Cd in the b-domain of metallothionein, Protein Engineering 1998 11(1);41–46
- Chen et al.: Hg2+ removal by genetically engineered Escherichia coli in a hollow fiber bioreactor, Biotechnology progress (1998) 14(5);667-71
- Chen & Wilson: Genetic engineering of bacteria and their potential for Hg2+ bioremediation, Biodegradation (1997) 8(2);97-103
- Deng et al.:Cadmium removal from aqueous solution by gene-modified Escherichia coli JM109 Journal of hazardous materials (2007) 139(2);340-4
- Deng et al.: Continuous treatment process of mercury removal from aqueous solution by growing recombinant E. coli cells and modeling study, Journal of hazardous materials (2008) 153(1-2);487-92
- Deng et al.: Bioaccumulation of nickel from aqueous solutions by genetically engineered Escherichia coli, Water research (2003) 37(10);2505-11.
- Fowler: Intracellular Compartmentation of Metals in Aquatic Organisms: Roles in Mechanisms of Cell injury, Environmental Health Perspectives (1987) 71;121-128
- Kao et al.: Biosorption of nickel, chromium and zinc by MerP-expressing recombinant Escherichia coli, Journal of hazardous materials (2008) 158(1);100-106
- Kostal et al.: Enhanced Arsenic Accumulation in Engineered Bacterial Cells Expressing ArsR, Applied and environmental microbiology (2004) 70(8);4582–4587
- Ngu & Stillman: Arsenic binding to human metallothionein, Journal of the American Chemical Society (2006) 128(38);12473-83.
- Singh et al.: Highly Selective and Rapid Arsenic Removal by Metabolically Engineered Escherichia coli Cells Expressing Fucus vesiculosus Metallothionein Applied and environmental microbiology (2008) 74(9);2924-7
- Yung-Fen et al.: ArsD Residues Cys12, Cys13, and Cys18 Form an As(III)-binding Site Required for Arsenic Metallochaperone Activity, The Journal of Biological Chemistry (2007) VOL 282(23); 16783–16791
- Yung-Fen et al.: ArsD: an As(III) metallochaperone for the ArsAB As(III)-translocating ATPase, Journal of Bioenergetics and Biomembranes (2007) 39;453-458
