Team:UANL Mty-Mexico/Project/capture

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iGEM UANL 2012


Capture


Arsenic capture is achieved by two proteins in our system: ArsR and rhMT. E. coli's ArsR has been previously overexpressed to develop a biological device for arsenic bioremediation  (Kostal et al. 2004). rhMT is a recombinant human metallothionein with an increased capability of arsenic capture -the highest known to date- able of binding up to 6 arsenic molecules (Ngu et al. 2006), in contrast with ArsR which can only bind one arsenic molecule. Thus rhMT will be the main chelating component of our bioremediator device.


Oligomerization


Oligomerization has been proved to enhance or reduce the function of some proteins (Ali and Imperiali 2005). ArsR is able to repress pArsR only as a homo-dimer (Wu and Rosen 1993). On the other hand, oligomerization of some metallothioneins results in an enhanced arsenic binding capability without compromising the protein's expression (Hong et al. 2000). We had chemically synthesized rhMT's CDS suitable for oligomerization ( K888007).


Hong et al. also observed that oligomerized metallothioneins form inclusion bodies.  To avoid such aggregation, solubility tags such as MBP and GST, are usually added (Hammartörm et al. 2002). We looked for the better suited solubility tag for our oligomer. DelProposto et al. (2008) reported a monomeric version of Mocr protein from T7 bacteriophage as a solubility tag, which together with MBP, yielded a better solubilization when compared to other common tags. Nevertheless MBP tends to form disulfide bonds that could interfere with metallothionein activity, given that its capture site is highly cystein rich. Thus Mocr will be used as the solubility tag for oligomerized rhMT. 


Transport


Arsenic is imported via passive diffusion. In order to increase its uptake, aquaglycerol porin GlpF will be used, which facilitates arsenic, glycerol and antimony import (Meng et al. 2004). Combined with the rhMT expression (Figure 1), we expect to obtain a system with increased arsenic chelating capability, considering that metallothioneins “neutralize” the charge and toxic effect of their ligand (Sigel et al. 2009).


Figure 1. Genetic circuits for arsenic capture and transport.


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