Team:MITADTBIO Pune/Design
PEred has been engineered to take on the plastic waste crisis, specifically that borne out of discarded sanitary napkins that comprise 90% plastic components. To do this, we have formulated a genetic circuit that includes a number of genes to do our dirty work.
The mode of pre treatment
According to our literature survey, deterioration of polyethylene works best by first treating with abiotic agents such as temperature or radiation, and subsequent action of microbial enzymes that modify and consume the polymer leading to changes in its properties. UV irradiation plays an extremely important role in our project, helping out in the following ways:
1. It is observed that oxidized groups by UV treatment modulate microbial attachment by increasing the hydrophilicity of the PE surface via photooxidative effect.[1]
2. It helps sterilize the used pads before exposing them to PEred as UV has a germicidal activity.
3. UV irradiation would also help us release ions from erythrocytes for effective uptake by the bacteria.[2]
The trigger mechanism
While brainstorming for what elements of the substrate to use as trigger, we narrowed down our choices to three components:
1. Using plastic as a trigger seemed futile. The plastic chains are so large that they can't enter the microbial cell.
2. The next element on hand is menstrual blood. Haemoglobin happens to be abundant in blood. We can use Fe2+ ions from haemoglobin degradation since bacterial systems have iron uptake channels. But according to our literature studies, most haemoglobin uptake/ degradation are present in enterohemorrhagic and pathogenic bacteria[3]. Cloning the enzymes required in the haemoglobin degradation pathway in our own host would render it pathogenic. This is neither safe for us nor for people who will work with PEred in future.
3. Next to haemoglobin, we have a lot of other ions involved in signal transduction mechanisms of the cell. One such ion, potassium, caught our eye. Therefore we decided to use potassium to trigger the circuitry of our genetically engineered bacteria.
Potassium uptake mechanism
Potassium is one of the vital elements for a living organism. It is required for key cellular
activities in prokaryotes as well as in eukaryotes.
We have chosen potassium as a promoter as it is an essential part of all living organisms. Thus
the bacterial species have three channels like Trk/Ktr/HKT, Kup/HAK/KT and Kdp to uptake monovalent potassium ion. Among the three, Kdp is an inducible promoter and the only transport system whose expression is regulated and induced by environmental potassium level and when the other transport system like Trk and Kup are nonfunctional.
Kdp-ATPase is a complex consisting of four subunits KdpFABC operon which encodes proteins responsible for stabilising the complex, binding as well as translocation of potassium by KdpF and KdpA proteins respectively. KdpB and Kdp C are associated with ATP hydrolysis. Kdp-ATPase system even consists of two component response regulatory KdpDE which
comprises of KdpD and KdpE proteins. Where KdpD is a Histidine kinase and KdpE is a response regulator[4]. This Kdp-ATPase system is well characterized in E.coli, M. tuberculosis,M. avium, M. bovis, M. marinum, and others except in M. leprae and M. ulcerans.
Potassium concentration, ionic strength and ATP content stimulate histidine kinase KdpD. After detecting these stimuli, KdpD experiences auto-phosphorylation, with its phosphoryl group transferred to the transcriptional regulator KdpE.
The phosphorylated KdpE (KdpE~P) binds to a 23-bp T-rich sequence in the promoter of kdpFAB and activates the expression of the transporter components KdpFABC.
Laccase: The hero of our project
Laccases are multi-copper enzymes that use molecular oxygen to oxidize various aromatic and non-aromatic compounds by a radical-catalyzed reaction mechanism. Laccases play a role in the formation of lignin by promoting the oxidative coupling of monolignols, a family of naturally occurring phenols. Other laccases, such as those produced by the fungus Pleurotus ostreatus, play a role in the degradation of lignin, and can therefore be classed as lignin-modifying enzymes.
Laccases have received much attention from researchers in the last decades due to their ability to oxidize both phenolic and non-phenolic lignin related compounds as well as highly recalcitrant environmental pollutants, which makes them very useful for their application to several biotechnological processes. [5]Such applications include the detoxification of industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, use as a tool for medical diagnostics and as a bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. In addition, their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes.[6]
PEred has been engineered to scythe down the plastic component of discarded sanitary pads, this being mediated by our star enzyme, laccase. For the purpose of laboratory level experiments and research, just secreting laccase would be sufficient, however, we needed to take into consideration the fact that PEred’s playground would comprise a massive amount of substrate in the form of discarded sanitary pads and menstrual blood. Herein lay a challenge. "How could we design PEred to be versatile enough to undergo healthy growth on its substrate whilst also secreting laccase to carry out the plastic breakdown?"
Using a Biofilm #MakeSense
Biofilms are a microbial product that allows bacterial cells to form colonies and adhere to a certain surface. This not only facilitates cell-cell interaction for enhanced metabolic cooperation and growth but also increases cell survival, defense and nutrient availability. The slimy polysaccharide layer characteristic of biofilm would hold PEred in good steady state, conferring it with an ability to not only adhere to the surface of the pads but also allowing it to spread across the entire surface of the pad it has to decompose.
Since we intended to use E. coli as the chassis around which to build PEred, it only made sense to exploit the biofilm-producing capabilities of microorganisms for our objective. The K-12 strain of E. coli has been studied extensively when it comes to biofilm production primarily via the secretion of curlifimbrae (adhesive fibers). The CsgABC and Csg DEFG operons as such are of primary concern.[7]
Inherently, biofilm production by E.coli is rather cryptic and not a constant process. For PEred to function suitably we’d need to be able to regulate its biofilm secretion. To do this, it was logical to use either the CsgD gene or OmpR gene in our genetic circuit being engineered.
While the Csg A and Csg B genes code for the actual structural components of curli fibers, the Csg DEFG Operon codes for accessory proteins necessary for proper curli assembly. Keeping this in mind, PEred has been engineered with the OmpR gene downstream of our K+ promoter. This would mean an augmented expression of OmpR which in turn would theoretically lead to an upregulation in CsgA and B expression and thus biofilm formation.
Where do we go from now?
Given all the information we have gathered from literature review and discussion, here is the proposed methodology of our genetic circuit.
