To execute our idea in real life scenario we first decided to construct a plasmid that can facilitate an extracellular expression of our protein of interest i.e. Alkyl sulfatase. We decided for extracellular expression as it provides several advantages over cytosolic production in terms of purity of recombinant protein obtained as a final product and less protease activity in the culture leading to more yield. Note, that we first tried building a device that would act as a template to which our protein of interest could be cloned.
It is difficult to accomplish efficient secretion of protein without trying a different combination of secretion tags and host-vector system. Thus, it was necessary that we choose different parameters for extracellular expression for sufficient production of our protein.
Fig. 1 Two modes of extracellular secretion 1) Secretion via protein with pelB secretion takes via transport of protein into periplasm via Sec pathway. 2) Secretion of protein via ompT secretion tag takes place by recognition of tag by ompT membrane receptor and excreting it outside the cell.
We decided to use two types of secretion tags for extracellular expression of our protein. PelB(pectate lyase B) secretion tag uses two-step sec pathway for secretion of the tagged protein into the periplasm of e.coli bacteria. And it has been reported that after a long time of incubation, cell permeability increases which lead to escape of the protein from periplasm. OmpT tag works in a different manner, in this, the protein is directly secreted out by the extracellular membrane protein that can recognize its secretion tag. Thus, cell integrity does not need to get compromised. (Fig. 1)
We also decided to optimize promoter strength to control the level of expression. Considering we want to keep economic investment in our system to the minimum we chose promoters which are constitutively expressed rather than promoter that require induction. To obtain a variable spectrum of promoter strength we choose Members of the Anderson promoter which are a collection of promoters suitable for general protein expression in E. coli.
We have cloned super folded Green fluorescent protein along with repeats of histidine tag downstream of PelB and ompT sequences in our construct which would serve a useful role for characterization of extracellular expression of our protein of interest. To facilitate cloning of our protein of interest we have included a site for the Nru1 restriction enzyme.
Fig. 2 Below is the basic layout of our BioBrick that are submitted in iGEM registry.
We were able to clone Anderson promoter Bba_J23100, BBa_J23102, BBa_J23105 upstream of pelB sequence and BBa_J23102, BBa_J23105 upstream of ompT tag. We have submitted three of these sequences as biobrick in iGEM registry.
Fig 3. Combination of different Anderson promoter with PelB and OmpT secretion tags.
We received plasmid from team Tec cem who were previously working on similar project to degrade SDS using alkyl sulfatase. We used the plasmid received from them to amplify SDS sequence using primers (figure). We have included a restriction enzyme site Nru1 in our device between secretion tag and sfGFP where we would insert the amplified sequence.
We also plan to increase our library for secretion tags by including signal sequence that translocates using twin arginine translocation system (TAT) which is a system that is Sec independent and capable of folding proteins better if they consist of twin arginine sequence.
Design of our Bioreactor
In order to implement our idea in natural settings we also created autocad model for implementation in households and directly in rivers. You can check them in Integrated Human Practices section of our wiki.