Overview
Our Project is divided into two distinct aspects. One the one hand, our goal is the removal of heavy metals from sludges. We managed to prevent lead agregation in Cupriavidus metallidurans CH34 to enhance its specificity for other metals, by using homologous recombination to mutate the transcription factor responsible for the main lead resistance operon transcription. On the other hand, we aim at producing ethanol from the cellulose contained in sludges. To achive that, we used external pre-purified cellulases to degrande cellulose int glucose, then we gave to our chassis (cupriavidus metallidurans CH34) the genes responsible for alcoholic fermetation to make it produce ethanol from glucose.
As our main chassis, among the many existing bacteria, we chose Cupriavidus metallidurans for three main reasons :
- it have naturally string resistance mechanism against a wide range of heavy metals
- these mechanisms are rather well documented
- it's genome is entirely sequenced and is widely antoted and avaiable for free on the internet.
Heavy metal resistance modification
Our idea was to modify the resitance mechanisms of the Gram necative bacteria Cupriavidus métallidurans to enhance or specify its heavy metal agregation.
To do that, we searched in scientific literature and eventually gathered some information relative to some of its heavy metal resistance mechanism pathways.
The figure above describe the lead resistance mechanism of Cupriavidus metallidurans encoded by the three pbr operons : lead seems to diffuse freely through the outer membrane. it enters the cytoplasm, with the help of the pbrT protein,
where it binds the pbrR proteins that thus acts as transcription factors and avtivates pbr operons transcription. Produced pbr proteins then contribute either to extrude lead (blue ones on the figure above) or to agregate metals (red ones).
To shut down the lead resistance, to specialize our Cupriavidus metallidurans for other metals, we chose to target the main pbr operon's pbrR activity. As transcription activator of the main pbr operon - responsible for lead agregation - its disapearence will lead to a non activation of the lead-agregation-related-proteins exprestion in favor of the extrusion mechainsm encoded by the two other pbr operons.
To break this mechanism, we have different options : target gene ORF, target promoter, target mRNA or traget protein. Again, after some investigation in the literture, we found out that a homologous recombination mediated gene deletion process is widely used in cupriavidus metallidurans CH34 and therfore we chosen this one to carry out our modifications in Cupriavidus metallidurans.
The figure above describe the principle of this strategy. 300bp upstream and downstream regions of the traget gene are amplified by PCR then integrated into the pCM184 plasmid on each side of a LoxP-bordered-Kanamycin-resistance-gene-ORF. Our Cupriavidus metallidurans is transformed with this plasmid, then, the homology between the pCM184 integrated inserts and the targeted gene borders enable homolgous recombination events. These resusts in a relacement of the target gene ORF with a kanamycin-resistance gene ORF. As this plasmid is not able to replicate in Cupriavidus metallidurans, it is quickly eliminated after the recombination events, through cell divisions.
ethanol
The other part of our idea was to produce ethanol from the cellulose contained in sludges. However, our chassis, Cupriavidus metalidurans CH34, does not have this ability.
Thus, we decided to provide it the enzymes necessary for alcoholic fermentation. These enzymes, alcohol deshydrogenase and pyruvate décarboxylase, well known in saccaromyces cerevisiae, are also naturally produced by a bacteria, Zymomonas subtilis.
Moreover, a previous igem team (edinburgh 2013) have already develped a fusion of this two protein from Z. mobilis, BBa_K1122673, with an enhanced efficiency compared to the individual ones.
However, two problems remained :
on the one hand, how to epress efficiency this fusion protin into C34. On the second hand, this stratgy allows to produce ethanol from glucose, not from cellulose. Therefore, we searched the literature for a broad range expresion vector prone to replicate in C34, an for a strong romoter in CH34.
thus, pBBR1MCS-2 appeared to be commonly used for expression in CH34, and the pan promoter, originally from Bacillus subtils, is known for being strong (even at without induction) and inducible by metals such as Cadmium or lead when use in E. coli or C. metallidurans. We then designed our expression vertor. To build the plasmid, we got pBBR1CMS2 from Addgene,
BBa_K1122673 part from iGEM and Pan from eurofins (de novo synthesis), then combined the different eement by using standard molecular biology technics (see experiments).
Concering the cellulose, we chose to use external pre-purified cellulases to process sludges before we use our bacteria.
References
[conjugaison] Wiesemann N, Mohr J, Grosse C, et al. Influence of copper resistance determinants on gold transformation by Cupriavidus metallidurans strain CH34. J Bacteriol. 2013;195(10):2298–2308. doi:10.1128/JB.01951-12
[pCM184] Broad-host-range cre-lox system for antibiotic marker recycling in gram-negative bacteria. Marx CJ, Lidstrom ME. Biotechniques. 2002 Nov;33(5):1062-7. 10.2144/02335rr01 PubMed 12449384
[pBBR1MCS-2] Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM 2nd, Peterson KM. Gene. 1995 Dec 1;166(1):175-6. 0378111995005841 [pii] PubMed 8529885
[pan] Ribeiro-dos-Santos, G., Biondo, R., Quadros, O. de F., Vicente, E. J., & Schenberg, A. C. G. (2010). A metal-repressed promoter from gram-positive Bacillus subtilis is highly active and metal-induced in gram-negative Cupriavidus metallidurans. Biotechnology and Bioengineering, 107(3), 469–477. https://doi.org/10.1002/bit.22820