Overview
The Engineering Cycle is a step-by-step method for developing and maintaining a biological system. It is used to identify problems and narrow down the concept to a single efficient prototype. The cycle begins with design, in which the team brainstorms an idea and draughts a model design. The next step is to build the prototype and bring the design to life. The third step is to put the prototype through its paces and check for errors and efficiency. The third step is followed by the learning cycle, which corrects any errors or inefficiencies. The cycle is repeated until the project is optimized with the final test run.
Module 1 : Parts Engineering
Iteration 1
Design
Our goal is to facilitate bioprecipitation of cadmium ions from industrial effluent for which the engineered bacteria should satisfy two major conditions. The bacteria should be able to form biofilm and it should express acid phosphatase enzyme. From the organisms on the white list cataloged by iGEM, we chose E.coli DH5 alpha and E.coli K12 MG1655 strains to check if it would fulfill the conditions[1]. Finally after literature mining and observing biofilm formation in both the strains, E.coli K12 strain was selected. The structure and other physiochemical properties of acid phosphatase enzyme were studied in detail[2]. Dephosphorylation of the substrate takes place when acid phosphatase binds with the phosphate substrate. Bioprecipitation takes place as the metal phosphate is not soluble in aqueous solution. We were introduced to curli biogenesis by Dr.Saravanan during the internal iHP meet. Then, we found out that the ability of the E. coli to produce biofilm is majorly dependent on csgA[3][4].Our initial plan was to modify BBa_K1404006 part submitted by the team INSA Lyon 2010 by linking acid phosphatase gene to csgA with the help of a rigid linker[5]. Thus, the fusion protein will be present attached to the curli of biofilm and will not get washed away by effluents.
Build
We chose the Biobrick RFC[10] standard as the majority of parts in the Registry database are RFC[10] compatible. Scar sequence for Biobrick RFC[10] was identified and used during virtual plasmid construction with the parts in Benchling. We had the opportunity to meet Dr.Anandan sir during this phase. He pointed out the fact that it would be difficult for the bacteria to achieve a curli- mediated transport of the phosphatase enzyme since the fusion protein shouldn’t affect the biofilm strength and he asked us to predict the fusion protein structure and check the binding strength of the fusion protein.
Test
To predict the structures and binding strength, we used I-TASSER .We computationally modelled the fusion protein (csgA aphA). Then,We received five predicted models from I-TASSER with a c-score in the range -4.7 to -3.2.
Learn
As suggested by Dr.Anandan during our iHP meeting, the result should be in such a way that the expression of the enzyme should not affect the biofilm production. We were working on E.coli K12 strain which is a poor biofilm forming strain. So the parts that we use in our project should enhance biofilm formation and express the enzyme at the cell surface.
Iteration 2
Design
From what we learnt, it is clear that the parts we choose must enhance biofilm formation and express acid phosphatase enzyme at the cell surface in the desired E.coli K12 MG1655 strain. Hence we planned to modify the composite part BBa_K2229300 submitted by the team iGEM17_TAS_Taipei by replacing the BBa_J23100 promoter with another strong promoter to reduce the burden on the organism and OmpR upregulation will facilitate csgD overexpression which in turn aids in curli biogenesis. This curli composite part BBa_K4509469 is joined with another composite part BBa_K4509669 consisting of a cadmium metal sensing promoter (BBa_K896008), strong RBS (BBa_B0034), cell surface tag (BBa_K103006) linked to acid phosphatase gene (BBa_K4509369), with a double terminator (BBa_B0015). This composite part has been designed to express the acid phosphatase enzyme on the cell surface.
Build
According to the design, the parts were constructed using Biobrick RFC[10] standard. We chose the RFC[10] standard because the curli composite part that we chose to modify followed the RFC[10] standard and the parts in the cell surface aphA composite part were all RFC[10] compatible. The scar sequence for RFC[10] was used. We were able to virtually construct the plasmid with the help of Benchling. We are being mentored by iGEM team member Deepak Kumar, who gave us insights about the standard and assembly methods and helped us in construction. OmpR upregulation facilitates csgD overexpression which in turn helps in curli biogenesis and the ompA-acid phosphatase fusion protein is expressed on the cell surface.
Test
To predict the structures and binding strength, we used I-TASSER .We computationally modelled the fusion protein (OmpA aphA). Then, We received five predicted models from I-TASSER with a c-score in the range -4.2 to -3.4.
Learn
As expected, the overexpression of csgD enhanced biofilm formation. From the information obtained by computationally modeling (I TASSER) the fusion protein, the c-scores of the fusion protein after modification have better scores when compared.
Module 2 : Reactor Engineering
Iteration 1
Design
The primary goal of project CURLIM is to create a method for removing cadmium from effluent. We intend to construct a bioreactor that facilitates the growth of genetically modified E. coli K12 to form biofilm, which will produce acid phosphatase. After that, the enzyme will dephosphorylate the phosphate monomers (substrates) to produce free phosphate. Cadmium phosphate will precipitate once the free phosphate bonds to the cadmium ions in the effluent. We are designing a laboratory Adaptable Moving Bed Biofilm Reactor in order to identify the key requirements for our user-level bioreactor and make design predictions (MBBR).
Dr. Venkat of the Madras Atomic Power Station in Kalpakkam gave the Team a unique perspective on Lab Scale Reactor Construction when they spoke with him. Dr. Anandan, Assistant Professor at Madras University's Tharamani Campus, also gave us constructive feedback on the environmental and reactor conditions to take into account, as well as the Enzyme Kinetics parameters. As a result, we realized that the Field Adaptable Moving Bed Biofilm Reactor (MBBR) needed to be as simple and configurable as possible so that end-users could build it with whatever resources they had available.
Build
Our design includes a cylindrical tank that will serve as the Primary Lab Adaptable Reactor Setup's primary culturing tank. The GAC OR plastic bio carriers will serve as a surface for the growth of biofilms. The first step was to drain the media solution after the main tank had produced the required quantity of biofilm, allowing the bio carriers to settle. The feed, which contains synthetic effluent, phosphate substrate, and microbe nutrients, is then added to the tank with the bio carriers. To maintain a consistent contaminant composition, synthetic effluent was created in the laboratory and used throughout the investigation. The main apparatus, which was kept, consisted of an aerator with membrane, a 4L cylinder, a 2-way peristaltic pump to control inlet and output flow, and a half L conical flask.
Test
There was an evidential growth of biofilm but it was inefficient. The secreted acid phosphatase enzyme interacted with the heavy metals in the feed, resulting in the formation of Cd-Phosphate complexes and sediment. Thus, bio-precipitation was performed in the MBBR.
Learn
The efficient formation of biofilms necessitates an optimum temperature of 37 degrees Celsius, which the primary adaptable reactor setup could not provide.
Iteration 2
Design
The team conducted additional research to optimize the environmental and reactor conditions. These investigations resulted in an improved version of the Laboratory Adaptable Moving Bed Biofilm Reactor (MBBR) which is divided into two parts. The enhanced setup incorporated a water bath thermal insulation layer outside the reactor tank to keep the temperature between 28 and 37 degrees Celsius, and another was an inner zone of the reaction site. Industries may effectively choose the best design that suits their demands by offering essential growing conditions and design recommendations. More information can be found on our Bioreactor page!
Build
A 4 L lab-scale batch reactor was constructed using commercially available Plexi glass vessels. It was split into two sections. One was an outside water bath thermal insulation layer to keep the temperature between 28 and 37 degrees Celsius, and the other was an inner zone of the reaction site. The air grid and influent pipes were installed at the reactor's bottom, as shown in Figure. Air was supplied from the reactor's bottom at a calculated flow rate of 1.8 m3/h to provide oxygen for the microbial mass's biological activity and to fluidize the carriers. Two separate peristaltic pumps are used to feed the influent to both reactors while maintaining their respective HRTs. To separate solids from effluent, two half-liter sludge settlers are built downstream of the reactors.
The biofilm carriers used were made of GAC (0.4-0.5 g/cc) or PE plastic (density 0.94-0.96 g/cm3) and were designed to provide protected surface areas for microbial biofilm development. The influent source was carcinogenic Cadmium-containing synthetic wastewater.
Test
There was significant growth of biofilm. The secreted acid phosphatase enzyme interacted with the heavy metals in the feed, resulting in the formation of Cd-Phosphate complexes and sediment. Thus, bio-precipitation was executed in the MBBR.
Learn
Laboratory Adaptable Moving Bed Biofilm Reactor (MBBR) that would simulate the conditions that industries would use to treat cadmium ions considering that this setup would later guide us in determining the necessary specifications for our user-level bioreactor as well as making design predictions.
References
1. Brombacher, E., Baratto, A., Dorel, C., & Landini, P. (2006). Gene Expression Regulation by the Curli Activator CsgD Protein: Modulation of Cellulose Biosynthesis and Control of Negative Determinants for Microbial Adhesion. Journal of Bacteriology, 188(6), 2027-2037. http://doi.org/10.1128/JB.188.6.2027-2037.2006
2. Evans, M. L., & Chapman, M. R. (2014). Curli biogenesis: order out of disorder. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1843(8), 1551-1558.
3. https://www.uniprot.org/uniprotkb/P0AE22/entry
4. http://2014.igem.org/Team:INSA-Lyon/Biology
5. http://2017.igem.org/Team:TAS_Taipei
6. Martinez-Hackert, E., & Stock, A. M. (1997). The DNA-binding domain of OmpR: crystal structures of a winged helix transcription factor. Chemistry & Biology, 4(2), 162. doi:10.1016/s1074-5521(97)90269-6
7. Olsén, A., Wick, M. J., Mörgelin, M., & Björck, L. (1998). Curli, fibrous surface proteins of Escherichia coli, interact with major histocompatibility complex class I molecules. Infection and immunity, 66(3), 944-949.
8. Reinke, A A, and J E Gestwicki. “Insight into amyloid structure using chemical probes.” Chemical biology & drug design., U.S. National Library of Medicine, June 2011, www.ncbi.nlm.nih.gov/pubmed/21457473.
9. Reisner, A., Haagensen, J. A., Schembri, M. A., Zechner, E. L., & Molin, S. (2003). Development and maturation of Escherichia coli K-12 biofilms. Molecular microbiology, 48(4), 933-946.
10. J. Xing, R. Hickey, Response in performance of the GAC-fluidized bed reactor process for BTX removal to perturbation in oxygen and nutrient supply, International Biodeterioration & Biodegradation, Volume 33, Issue 1,1994, Pages 23-39, ISSN 0964-8305, https://doi.org/10.1016/0964-8305(94)90053-1.
11. Magdum, Sandip & V., Kalyanraman. (2019). Evaluation of High Rate MBBR to Predict Optimal Design Parameters for Higher Carbon and Subsequent Ammoniacal Nitrogen Removal. Current science, 116. 2083-2088. 10.18520/cs/v116/i12/2083-2088.
12. Brackin, M.J., McKenzie, D.E., Hughes, B.M. et al. Laboratory-scale evaluation of fluidized bed reactor technology for biotreatment of maleic anhydride process wastewater. Journal of Industrial Microbiology 16, 216-223 (1996). https://doi.org/10.1007/BF01570024.
13. Yu N, Zhang D, Lei Y, Wang J, Dong Y, Chen Y. Innovative Coating-Etching Method of Biocarrier Fabrication for Treating Wastewater with a Low C/N Ratio. Polymers, 2022 Jul 25;14(15):3010.