Team:IISc-Bangalore/Proof Of Concept
Proof of Concept
To demonstrate the actual potential and feasibility of our concept, we went much further than envisioning ideas and researching scientific literature to support them. We started working in the lab to aim for conclusive results, which show the success of the different components of our system. We want to showcase an example that affirms the potential of our project. Here, CellOPHane gets into action, and emphasizing its feasibility is the key.
Engineering and implementing the whole workflow in relation to the proposed hardware is extremely challenging. Thus, we focused on developing a proof of concept for our novel bioremediation platform.
Planned Workflow
First of all, we want to express as well as purify C1 and C2.
Then, we wish to check the binding affinity of C1 to bacterial cellulose surface.
Then, we want to probe into the mutual affinity between C1 and C2.
Next, we intend to characterize the binding of C1-C2 adduct on BC surface.
Finally, our target is to check the efficiency of this functionalized filter sample in cleaving organophosphate substrate(s) by performing (mainly spectrophotometric) assays.
Progress
We have successfully expressed both C1 and C2.
Though C1 is soluble and ready to undergo the purification protocol, we failed in C2 pulldown even after using many methods. Even C2(2.0) could not definitively be proved to be soluble, just like C2 itself.
We observed that the beads used in C2 pulldown turned green (almost surely due to sfGFP) each time. However, the subsequent SDS-PAGE did not show the expected POI (i.e. protein of interest) band. Interestingly, the band corresponding to sfGFP alone was visible! A probable explanation behind this issue is that C2 got cleaved between OpdA and sfGFP, and the OpdA (quite hydrophobic) underwent proteolytic cleavage. The expression host might get benefitted from this, given that OpdA is prone to aggregation. If OpdA was really facing post-expression proteolytic cleavage, it would imply our inability to go beyond the first step of the Proposed Workflow under the current design. If it comes out to be true, the success of CellOPHane is bound to get delayed - very delayed!
Time was scarce and pandemic was posing a new challenge before us every single day. Thus, we planned to take a detour. We set out to see whether OpdA is getting degraded inside the expression host or not. This is why we grew our expression host in an alternative media (i.e. Terrific Broth or TB) in addition to Luria-Bertani Broth (i.e. LB), performed Bradford protein assay on the crude extract of the large-scale secondary culture, followed by the OpdA activity assay on the same. Lysates of EV (empty vector; expression host transformed with only pETMCN-T7) and NT (non-transformed; expression host without any inserts) cultures were used as the necessary controls
We normalized the absorbance of the test samples at 348 nm (corresponding to λmax of chlorferon, the degradation product of assay substrate coumaphos) with their respective protein concentrations as deduced from the Bradford assay. In OpdA assay, NT was used as the blank (thus, futile to deal with its absorbance, be it normalized or not). The normalized absorbance (in arbitrary units) values are tabulated below:
| Culture Medium | EV | C2 | C2(2.0) | NT |
|---|---|---|---|---|
| LB 1 | -0.31834 | 0.20386 | 0.34964 | Used as Blank |
| LB 2 | 1.38791 | 0.57668 | 0.16738 | Used as Blank |
| Average of the values | 0.53479 | 0.39027 | 0.25851 | - |
| TB 1 | 0.37180 | 0.82515 | 4.38322 | Used as Blank |
| TB 2 | 2.05646 | 1.58143 | 1.87226 | Used as Blank |
| Average of the values | 1.21413 | 1.203 | 3.1277 | - |
Here '1' and '2' simply denote two replicates of the OpdA assay with the samples mentioned, in chronological order.
To understand the trend exhibited by the average values, a bar chart was plotted:
From this chart, we gain a number of valuable insights. Though it needs solid proof, we can propose a hypothesis. Thanks to the signal peptide sequence, C2 gets translocated from cytoplasm to periplasm. When E. coli is grown aerobically in LB, cultures become more alkaline during the log phase due to metabolism of amino acids. This high pH can accelerate the aggregation of C2 molecules in the periplasm. When host's extracytoplasmic stress response machinery cannot keep pace with the aggregation, the host loses vitality. Thus, normalized absorbance of C2 sample is lower than EV sample. Propensity of C2(2.0) forming aggregates under high pH is probably more than C2, leading to the even lower read-out for the C2(2.0) sample. TB media is known to increase the plasmid yield by extending the exponential phase of E. coli. Thus, we get higher amount of readout, in general, compared to the LB case. C2 inherently suffers from misfolding and thus C2 sample shows a decrease in the read-out compared to the EV sample. TB contains glycerol, which can be used by the cells as a carbohydrate source, while LB does not have additional carbohydrate, relying almost entirely on amino acids as the source of carbon and energy. E. coli metabolizes glycerol to produce organic acids (e.g. acetate), which is not as detrimental to the stability of C2(2.0) as excess alkalinity. Therefore, C2(2.0) sample contained a lot of functional C2(2.0) molecules, which led to the highest normalized absorbance among all.
This also showcases the fact that activity of the protein of interest is closely linked to the condition which expression host is grown in. The dramatically high read-out for C2(2.0) sample from TB media shows that OpdA mainly stays undegraded under this set of conditions. Interestingly, this holds irrespective of the validity of our hypothesized explanation.
The result empowers us to continue with the rest of the steps in our Proposed Workflow. It proves that our expression host can efficiently produce OpdA (and related fusions), which is the cornerstone of CellOPHane, provided the growth conditions are optimized. This piece of Proof of Concept strengthens our belief that CellOPHane is not a distant dream anymore.
References
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