Team:UM Macau/Experiments
Experiment Design
Design Overview
Our project aims to engineer an Escherichia coli that is capable to capture nanoparticles, activate itself and be able to be controlled through magnetization. Our efforts are to demonstrate the proof of concept that a controllable cleaner microorganism is possible. In the future, teams can use our results as a basis for a controllable wastewater cleaning organism that is safe and cheaper to mass produce for the application of helping improve water cleaning efficiency.
This humble project demonstrates the first step into developing a wastewater cleaning organism. A proof of concept, our engineered) E.coli uses a sticky protein on the cell surface called L-Dopa; which would be derived from tyrosine, in order to capture nanoparticles as our first feature. As a second feature our organism would also be able to secrete a functional tyrosinase enzyme in order to convert tyrosine into L-Dopa, which is the primary domain wherein nanoparticles or other suspended particles could bind into. As a third feature, we would demonstrate that our E.coli is magnetizable through the overexpression of FtnA which could store iron ions and make our E.coli magnetizable. Hopefully with our organism able to capture nanoparticles we are able to reduce the nanoparticle concentration in the suspended solution during the clarifier stage of the wastewater treatment plants.
Our Self Activating Nanoparticle Collector E.coli would be constructed in three phases as shown in Figure 1. First we would construct the plasmid which would contain our different composite parts (BBa_K3033018, BBa_K3033019, BBa_K3033020, BBa_K3033021, BBa_K3033022, BBa_K3033023 and, BBa_K3033024). We would primarily use the E.coli strain BL-21(DE3) which is a suitable E.coli strain for protein expression due to it being deficient of proteases Lon and OmpT which might affect our protein integrity. [7]
Figure 1. General workflow of the project.
Figure 2. Overview structure of the surface capturing system.
Our surface nanoparticle capturing protein (SNCP) has a couple of components as shown in Figure 2. An anchoring protein. A linker enzyme. Next is the presence of 6-histidine tag. Finally, the capturing domain. Our inspiration for this capturing system is from the Mefp-5 protein that is found on the foot of the mussels. This inspiration protein has L-Dopa and histidine repeats in them thus we want to express L-Dopa as our capturing domain as it has a sticky property.
Our system would primarily be anchored onto the cell membrane of the bacteria using the outer membrane protein called OmpW derived from E. coli itself, it forms an 8-stranded beta-barrel with a long and narrow hydrophobic channel. We chose this as our anchoring protein as it is very stable and is able to carry our other protein onto the surface of the bacteria. We have truncated this version of OmpW at the 191st amino acid as it has been shown to be stably anchored onto the cell membrane while being able expose the rest of the proteins onto the outside of the cell membrane. [1]
Figure 3. Structure of OmpW [2]
The next component is the Perhydrolase which was derived from the organism Pseudomonas aeruginosa. According to our adviser, this would be enhancing our capturing system’s capability thorough the process of epoxidation. The next component is the 6- histidine tag which would be used to check the protein expression.
Figure 4: Variants of our capturing system domains
Finally, we have the capturing domain wherein we have 4 different variation as shown in Figure 4. 2-Tyrosine, 6-Tyrosine, Mefp-5, and 2-Aspartic acid. We decided to modify the capturing domain to see whether which variant would be more effective at capturing nanoparticles. The 2-Tyr variant was chosen as it has been shown to be able to be converted to L-DOPA and bind to many surfaces, we used this variant as the baseline and modified accordingly.[1] We have added more tyrosine to see whether the addition of more tyrosine would further enhance the capturing function. Mefp-5 protein from Mussels itself was also used as a variant to compare the effectiveness of our other variants. 2-Aspartic acid was chosen due to a hypothesis the carboxyl group of found in the capturing domain is actually the main reason for the sticky function. In all of these variants except for Aspartic acid, would require the conversion of Tyrosine into L-Dopa using the tyrosinase which would be our second feature.
Figure 5: Biobrick design (Variations of OPHT part)
The design of our constructs for the capturing system are as shown in figure 5. We used a T7 promoter (BBa_K3033000) to drive the expression of our whole fusion protein. Then a Ribosome binding site (BBa_K3033002) in order to translate our fusion protein. It is then followed by the anchoring protein (BBa_K3033004) then the perhydrolase (BBa_K3033005) as a linker an enhancer of our sticky function through epoxidation. A histidine tag (BBa_K3033006) was put for later checking of protein expression using western blot. Our capturing domain which has 4 variants as shown earlier: OPHT with 2-tyrosine (BBa_K3033007), OPHTIII with 6-tyrosine (BBa_K3033008), OPHM with Mefp-5 (BBa_K3033010), and OPHA 2-Asp (BBa_K3033011). We also have made a variant of OPHT and put an eGFP tag on the N-terminal of the protein and thus we have made the OPHTII construct for an easy reporter checking the successful induction of E.coli and checking the localization of our protein. Finally, we have put a transcription terminator at the end which was derived from the pET11a plasmid (BBa_K3033015) so that the bacteria would not be wasting energy. Our capture protein fragments would be ligated into the pet11a plasmid backbone.
Tyrosinase is an oxidase which we would be using to synthesize L-DOPA on the cell surface of our engineered organism. We are using this enzyme to convert our expressed L-tyrosine on the capturing domains of the surface capture protein to L-DOPA. The chemical is shown in Figure 6a below. We have used a tyrosinase coding sequence (BBa_K3033013) which was derived from the tyrosinase producing bacteria called Bacillus megaterium strain M36. [3]
Figure 6. A: Chemical reaction where L-Tyrosine is converted to L-DOPA through the hydroxylation of the monophenol domain into o-diphenols. [6] B: The Sec-A dependent pathway for secretion of our protein of interest. [5]
Our second feature is a tyrosinase secretion system. This is a necessary system to have for the whole idea of our project due to the inability for L-Dopa to be synthesized naturally by E.coli. Therefore, the modified E.coli would be secreting an active form of tyrosinase into the supernatant where our organism would be located in, so that it would have contact with our capturing domains and convert tyrosine to L-Dopa. We plan to use the NSP4 signal sequence to carry our protein of interest to extracellular environment [4] Through the Signal Recognition Particle mediated sec-dependent pathway A as shown in Figure 6b above our protein would be secreted to outside of the cell. [5]
Figure 7. The composite part for our second feature, tyrosinase secretion system.
Our secretion system is designed as shown in, figure 7. We have constructed the secretion system to be under the araBAD promoter (BBa_K3033001), we would use the ribosome binding site (BBa_K3033003) to initiate the translation of the tyrosinase protein. 6 bp after the ribosome binding site we have put the secretion signaling peptide NSP4 (BBa_K3033012) as a signaling tag for our coding sequence of the Tyrosinase enzyme which was derived from Bacillus megaterium (BBa_K3033013). We then tagged our protein with 6-Histidine (BBa_K3033006) for later protein expression check. A rrnB T1 Terminator (BBa_K3033016) is derived from the pBAD24 plasmid to stop the transcription of unnecessary nucleotides to avoid taxing the cell’s machinery.
Figure 8. Surface capturing and tyrosinase secretion and activation system.
As shown in Figure 8 above, the first step is to express the capturing proteins on outermembrane. The second step is to secrete the active form of tyrosinase, onto the supernatant wherein our capturing system is exposed, then it would react with the capturing domain to convert L-tyrosine into L-DOPA. After this second step, the capturing system’s capturing domain is now ready to bind onto our nanoparticle targets.
The precise spatial arrangement of cells has been one of the most important topics in synthetic biology, and this feature also plays important role in our project, SANCE. After the surface capturing system is activated by self-secreting tyrosinase and capture nanoparticles by the adhesive fusion protein on its surface, the E.coli – nanoparticles complexes should be removed from the wastewater treatment system to complete its mission. Hence, we designed our E.coli to obtain the third feature of our project, the magnetization. With this feature, our E.coli could be magnetized and be removed away from wastewater treatment process when a magnetic field is applied as shown in Figure 9.
Figure 9: We aim to make our E.coli magnetizable so that we are able to collect them when they are applied to any waste water tank and extract them.
We accomplished this feature by cloning and overexpressing FtnA, which encodes for a globular protein complex called ferritin, consists of 24 protein subunits to form a nanoshell to store iron molecules by various metal-protein interactions ("Ferritin protein nanocages—the story"), Figure 10 shows the protein structure. Therefore, with the overexpression of FtnA, more ferritin proteins can help E.coli to store more Fe3+ ions within cytosol. [8] We expect that the magnetization of E.coli could be achieved within 30 minutes to 1 hour in real time observation. Thus, the removal of E.coli – nanoparticles complexes in wastewater treatment could be done within short duration.
Figure 10: The protein structural feature of E.coli ferritin. [9]
Our team has overexpressed the FtnA using the composite part BBa_K3033024 as shown in Figure 11 which would be ligated into a pBAD24 vector. The composite part is made up of the following components: (BBa_K3033001) which is the pBAD promoter, (BBa_K3033003) which is the ribosome binding site that is based on the pBAD24 plasmid, (BBa_K3033014) is the FtnA coding sequence gene which is derived from the E.coli organism, (BBa_K3033017) which is for the flag tag for later analysis of protein expression by doing western blotting, (BBa_K3033016) is the pBAD24 derived terminator for stopping the transcription of unnecessary nucleotides.
Figure 11: Composite part for the overexpression of FtnA, the gene is under the promoter pBAD and the Arabad RBS.
Reference
- Park J. P., Choi M.J., Kim S. H., Lee S. H., Lee H. (2014). Preparation of Sticky Escherichia coli through surface display of an adhesive Catecholamine Moeity. Applied and Environmental Microbiology. 80(1) p.43-53.
- Hong H., Patel D. R., Tamm L. K., Berg B. V. D. (2005) The Outer Membrane Protein OmpW forms an Eight-stranded beta-Barrel with a Hydrophobic Channel. The journal of biological chemistry. 281(11) pg. 7568-7577.
- Arikan B., Valipour E., (2015). Increased production of Tyrosinase from Bacillus megaterium strain M36 by the response surface method.
- Han S.J., Machhi S., Berge M., Xi G., Linke T., Schoner R. (2017). Novel signal peptides improve the secretion of recombinant staphylococcus aureus. Alpha toxinh35L in Escherichia coli.
- Bechkwith J., (2013). The Sec-dependent pathway. Microbiology. 164 pg 497-504
- Agarwal P., Singh M., Signh J., Singh R. P., (2019). Microbial Tyrosinases: A Novel Enzyme, Structural Features, and Applications. Applied Microbiology and Bioengineering. pg 3-19.
- Muhlmann M., Buchs J., Noack S., Forsten E.(2017). Optimizing recombinant protein expression via automated induction profiling in microtiter plates at different temperatures. Microb Cell Fact. 16(1), pg 200.
- Bitoun J. P., Wu G., Ding H., (2008). Escherichia coli FtnA acts as an Iron Buffer for Re-assembly of Iron- Sulfur Clusters in Response to hydrogen Peroxide Stress. Biometals. 21(6) 693-703.
- Image from the RCSB PDB (rcsb.org) of PDB ID 1EUM (T.J. Stillman, P.D. Hempstead, P.J. Artymiuk, S.C. Andrews, A.J. Hudson, A. Treffry, J.R. Guest, P.M. Harrison) (2001) The high-resolution X-ray crystallographic structure of the ferritin (EcFtnA) of Escherichia coli; comparison with human H ferritin (HuHF) and the structures of the Fe(3+) and Zn(2+) derivatives. J.Mol.Biol. 307: 587-603.)