Part:BBa_K2868015
HHTC-Re-CBD x2 Copper Binding
This is a fusion protein formed with a Chitin Binding Domain (CBD) and semi-rationally designed copper binding domain (HHTC-Re) as subunits. There are two metal binding domains, allowing for two copper atoms to be bound to each protein molecule. GSGGSG linkers were added in between the CBD and HHTC-Re subunits to spatially isolate the individual domains while still allowing for flexibility.
We also developed 3 fusion proteins: CBD-2xHHTC, CBD-3xHHTC, and CBD-6xHHTC for use in the actual filter, comprised of a CBD, and HHTC metal binding repeats interspaced with a GSGGSG flexible linker. These constructs also included a Lumio tag for downstream verification of protein production, and a 6x His tag for protein purification. A challenge incurred when designing these parts was that the His tag maintains some affinity for metals, including copper. This would interfere with downstream modeling and experimental analysis of the interaction between copper and the HHTC domain. We therefore added the Mxe GyrA Intein between the Lumio/His tag and the rest of the protein, as this intein could be cleaved through addition of 50 mM DTT through thiol-mediated cleavage after protein purification [19]. After DTT was added, we were able to obtain the fusion proteins ready for downstream application (as per Figure 2).
Figure 2: Schematic of the design of the fusion proteins (HHTC-Re x n) and the method in which they bind copper.
We also produced an RFP-CBD fusion protein to visualize the distribution of chitin on the surface of a piece of mycelium, as well as a proof of concept for aesthetic design purposes.
2.5 Wet-lab Experiments: Fusion Protein Production
Once all three constructs were ordered (IDT gBlocks), we ligated the linear DNA constructs to the PSB1C3 iGEM backbone using Gibson Assembly, transformed NEB T7 competent E. coli with our new plasmid, and plated the colonies on chloramphenicol selective LB plates. The colonies were incubated at 37℃ overnight or until there were distinct, visible colonies, and never longer than 72 hours. The existence of our DNA constructs in the colonies was confirmed using verification primers (VF2 and VR) in a colony PCR. We then performed His-tag purification on crude cell extract from our colonies using Thermo Scientific HisPur Ni-NTA spin columns. Following protein purification with the spin columns, we confirmed the presence of our protein in the final elution using Thermo Fisher Scientific’s Lumio Tag Detection Kit [20]. The standard protein purification protocol was modified by introducing a buffer containing 50 mM DTT for on-site cleavage, so the desired fusion protein could be eluted with the His-tag removed. We then performed a BCA Assay to determine our total protein concentration in each elution.
2.6 ITC Testing
Isothermal titration calorimetry (ITC; MicroCal iTC200) was employed to determine the association equilibrium constant (Ka), enthalpy (ΔH), and the number of ions bound per ligand (n). Ka describes the affinity of a ligand for its substrate, and we used it to quantitatively characterize the interaction between our peptide and copper [21]. All binding parameters for the test were within the specifications determined by the manufacturer. We used 10 mM, 2-(N-morpholino)-ethanesulfonic acid (MES) buffer for testing because it does not cause metal ion interference, and has a stable pKa over a wide temperature and pH range. Experiments were conducted at pH 5.5 to prevent copper precipitation, and pre-made copper stock solutions of known concentration were used.
Peptides were prepared for ITC by dissolving lyophilized protein (powder) in MES buffer, and ITC experiments were run at 25°C and set to deliver 20, 0.5 – 1 µL injections of Cu at 150 second intervals. The metal solution in the syringe was titrated into the peptide solution in the cell, and interactions were measured. Raw data were corrected by subtracting the heats of dilution, and collected data were fit with a one-site binding model using the Origin-7™ software.
Figure 4: Quark ab initio model of 2x-HHTC-Re with chitin binding domain. Domains within the fusion protein have been annotated to display the conformation and spatial orientation.
After confirming the copper binding of the individual HHTC-Re x n peptides, we then needed to assess whether our fusion protein could bind copper and chitin, and whether it could do so when already saturated with the other substrate. Figure 5 depicts two experiments a) Raw data and b) isotherm for 2x-HHTC-Re-CBD + NaDg and Cu. In this experiment, N-acetyl D-glucosamine (“NaDg”; analogous to a chitin monomer and widely used in the literature for assessing chitin binding) was first titrated into 2x-HHTC-Re-CBD and no isotherm was calculated because binding sites were not saturated by the ligand. Cu was then titrated into the 2x-HHTC-Re-CBD + NaDg complex and this resulted in a Cu affinity at Ka= 7.61 +/- 1.49x 106 M-1 that is largely comparable to 2x-HHTC-Re (no CBD) and lowerby an order of magnitude than 2x-HHTC-Re-CBD without bound NaDg. Data show 20 1 µL injections. 2x-HHTC-Re-CBD was selected as the candidate for testing because it displayed the most consistent and strong results during protein purification procedures, and seemed most promising for downstream applications (such as our filter). Work is in progress on optimizing the 3x and 6x fusion proteins and creating prototypes (thus far, protein production as been successful).
Figure 5: Isotherm and data produced by ITC for assessing the binding affinity of our fusion protein (2x-HHTC-Re-CBD) for chitin (represented by N-acetyl D-glucosamine) and Cu.
Tangential Flow Prototype Creation: Material Properties and Design Considerations
The first consideration when thinking about creating a mycelium biofilter had to do with the qualities and characteristics of the mycelium material. Would it be waxy? Would the chitin be exposed? Such questions and more could significantly impact the approach we picked for functionalizing any material produced. The first test we did was comprised of a simple dye being poured on a piece of mycelium to observe the hydrophobicity of the material and whether it could be easily penetrated. <p>It became evident that the mycelium surface is highly hydrophobic. While this may appear to be an undesirable trait for a water filter, we found that subsequent incubation of mycelium in moving water for a period of 48 hours allowed for the material to become permeable. In an interesting twist of synergy, the fusion protein we were attempting to produce was highly hydrophobic as well, and therefore probably showed good affinity for the mycelium material (Figure 6).
Figure 6: Biochemical properties and hydropathy of a CBD-HHTC-Re fusion protein. The fusion protein displays significant hydrophobic tendencies. While this was initially thought to be a problem, it synergized well with the nature of the mycelium surface and most likely allowed the protein to obtain a proximity close enough for chitin binding to occur. The simulation to produce the graphs above was done by <a href="https://www.lifetein.com/">LifeTein.</a>
Tangential Flow Prototype Testing: Bulk Surface Adsorption Using Phen Green
The Cu concentration in the initial copper solution was 325 (+/-25) µM Cu. After 30 minutes of tangential flow, interestingly, the untreated mycelium absorbed about 23% of the copper in solution, revealing a fascinating synergy in that the mycelium possesses some inherent metal sequestration properties. The treated mycelium (filter prototype) was able to sequester ~92% of the available Cu in solution - validating its efficacy and potential utility.
Figure 7: Cu (µM) remaining in solution for n = 3 samples after incubation for 30 minutes while undergoing tangential flow on a flatbed shaker (figure design credits: Jesica Urbina). Experimental conditions were maintained as described previously.
We also tested the filters over a period of 72 hours and measured the copper concentrations at the end in each of the three experimental trials. The filter was able to bind nearly all of the copper in solution to almost undetectable levels, and the plain mycelial material displayed remarkable properties as well.
Figure 8: Cu (µM) remaining in solution for n = 3 samples after incubation for 72 hours while undergoing tangential flow on a flatbed shaker (figure design credits: Jesica Urbina). Experimental conditions were maintained as described previously.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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