Team:TecCEM/Contribution

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Contribution

TecCEM Characterization, colorimetric assay and purification

For the characterization of the Laccase BBa_K863010 we conducted an IPTG induction experiment in which we used the transformation of the Laccase in E. coli BL21. We thought that we could use another strain called SoluBL21 but results were not successful as no expression was found. We verified the presence of the protein through an SDS-PAGE with a gel concentration of 12% and found a visible band with a mass of around 50 kDa. This can be seen in figure 1. 

Figure 1. The lanes correspond to the following. M: Molecular weight marker; 1: Total protein after induction; 2: Total protein before induction; 3: Protein found in the Culture Medium after induction; 4: Cytoplasmic soluble fraction; 5: Inclusion bodies of the insoluble fraction; 6: Concentrated Culture Medium after induction. The band observed in lanes 1, 3 and 6 weighs around 48 kDa and corresponds to the expected size.

Figure 2. In section a) we can see the change of color of the substrates used (methylene blue, malachite green and rose bengal from left to right). a)-I. corresponds to the cytoplasm soluble fraction while a)-II. corresponds to the secreted protein from the culture medium. In section b) we can see that the pH remained unchanged throughout the assay.

We found that the protein was mostly found on the culture medium but can also be found on the cytoplasmic soluble fraction. The band that was appreciated in figure 1. indicates that there’s an expression of the Laccase after it’s induction with IPTG so our results and experience using this part was different from what 2019 PuiChing Macau’s team reported previously.

 

To prove that the Laccase was being expressed, we conducted a colorimetric assay involving three colorants that act as a substrate: methylene blue, malachite green and rose bengal. We based this experiment upon the findings of D. Singh et al. (2014) [1], in which they used agar plates with these colorants to determine the expression of Laccases in a medium. The first assays we conducted were only to find out if there was any color change with the presence of our extracted Laccase either from the cytoplasmic soluble fraction or from the culture medium. We used Citrate Buffer and found a change of color in different samples of Laccase after its purification using Ni Affinity. We also verified that the effect we saw wasn’t related to a change in pH. The results are available in figure 2.

After this first assay, we decided that we had to establish a purification protocol through which we could get the most enzyme possible. We used a Ni Affinity Column and a system of recollection of the different phases. We collected the enzyme from both the culture medium and the cytoplasmic soluble fraction and measured the absorbance of the fractions collected at 280 nm. In total, we got 21 fractions for the cytoplasm proteins and 20 fractions for the culture medium. The purification conditions were established using a growing elution buffer concentration. These conditions are shown in figures 3 and 4.

Figure 3. Chromatogram of the purification of the cytoplasmic soluble fraction showing the spike of absorbance in an elution volume of around 15 mL to 20 mL (with a percentage of elution buffer of around 30 to 50%); corresponding to the fractions containing the Laccase.

Figure 4. Chromatogram of the purification of the culture medium showing the spike of absorbance in an elution volume of 12 mL to 20 mL (with a percentage of elution buffer of around 16% to 50%); corresponding to the fractions containing the Laccase.

Finally, we conducted a last assay in which we first quantified the amount of protein recovered through a BCA quantification protocol. Using this protocol and with the elaboration of a BCA curve, we estimated that we recovered 0.334 µg/mL of Laccase in the culture medium while for the cytoplasmic soluble fraction we obtained 0.1298 µg/mL of Laccase. This was consistent with the results we got from the chromatograms. 


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For the final colorimetric assay we conducted, we quantified the activity of the Laccase obtained from the purification. We compared it with a Commercial Laccase from Sigma belonging to Trametes versicolor and used the spectrophotometer to measure methylene blue, malachite green and rose bengal at 664, 617 and 562 nm respectively. Since we didn’t quite have the exact concentration of colorants in our samples, we limited to measure the activity as a percentage of degradation of each colorant where a 100% of substrate would be the absorbance of the control of the blue, green and rose colorants and the enzymatic degradation would be expressed as the loss of color. The results can be seen below.

Figure 5. Percentage of degradation of Methylene Blue through time for the Laccase in the culture medium (blue), soluble fraction (orange), purified culture medium (yellow), purified soluble fraction (light blue) and the Laccase from Trametes versicolor (gray).

Figure 6. Percentage of degradation of Malachite green through time for the Laccase in the culture medium (blue), soluble fraction (orange), purified culture medium (yellow), purified soluble fraction (light blue) and the Laccase from Trametes versicolor (gray).

Figure 7. Percentage of degradation of Rose Bengal through time for the Laccase in the culture medium (blue), soluble fraction (orange), purified culture medium (yellow), purified soluble fraction (light blue) and the Laccase from Trametes versicolor (gray).

In the assays, we saw a similar trend for the degradation of Methylene Blue. For Malachite Green, the Laccase from Trametes versicolor had a higher degradation rate. Finally, for Rose Bengal we observed a similar trend between Trametes versicolor Laccase and the Soluble Fraction Laccase (before purification). We then reported the percentage of degradation of each sample for each colorant at the final point in time and got the next results:

Figure 8. Percentage of degradation of each Laccase at the final point in time for each colorant.

We observed that overall, the best results were obtained by the Laccase of Trametes versicolor (as expected) followed by the soluble fraction and the purified soluble fraction. However, it is worth noting that although these results show that the commercial Laccase may have higher degradation values, it also has a higher concentration since it was prepared at 1 mg/mL (compared to the purified Laccases which had concentrations of 0.334 µg/mL in the culture medium and 0.1298 µg/mL in the soluble fraction. 

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Finally, we ran some experiments in which we tried to predict the structure of the protein so that other teams could use it to test the affinity of the Laccase towards any given substrate through molecular Docking. We uploaded the amino acids sequence of the enzyme to I-TASSER, a server which runs a platform that predicts the structure of a protein and its function through a model [2]. We compared the model we got with a Laccase from Trametes versicolor we got from PDB under the code 1KYA [3] using a structural alignment (seen in figure 9). 

Figure 9. Structural alignment of the model of the Laccase from BBa_K863010 predicted using I-TASSER (Light Brown) and the Laccase  1KYA from Trametes versicolor obtained from PDB (Light Blue). Image visualized using Chimera [4].

The model for the Laccase can be downloaded here.

3D printing: Developing a quartz crystal microbalance for future iGEM teams.

We developed a low-cost quartz crystal microbalance (QCM), compared to those sold commercially on the internet. We designed the housing to protect all electronic components and to have a visually appealing model. We use a software specialized in CAD (computer-aided design), which is Fusion 360, it handles a version for students and its use is simple, compared to other more complex ones. The process consisted of making measurements of our components and estimating the volume that would be necessary to house them within our microbalance. The objective is that it be able to be replicated by any iGEM team, for which we show below the plans with their respective measurements for the 3D printed parts. Check it here

Figure 10.  Technical drawing measures for microbalance first piece

In our case, we used a Zortrax model M200 3D printer with 1.75 mm thick ABS filament. The main case is shown below (Figure 11).

Figure 11. 3D printed pieces.

If you prefer, you can directly print the pieces, which are in "stl" format ready to be sent to the 3D printer. Such pieces are stored in this folder.

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In the same way, the exploded view is shown in figure 12, along with the main components required and their quantity.

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Finally, the easiest way to explain the assembly and how to replicate it successfully is through a video, which was also made with the help of Fusion 360.

Figure 12. Exploded View

If you are familiar with Fusion 360, you can create a copy of the complete assembly and make any modifications you require at the link  https://a360.co/2Yykcky

  • [1] D. Singh et al., “Isolation, Characterization and Production of Bacterial Laccase from Bacillus sp”, 06 2014, bll 439–450. 2014.
     

  • [2] A. Roy, A. Kucukural, en Y. Zhang, “I-TASSER: a unified platform for automated protein structure and function prediction”, Nature Protocols, vol 5, no 4, bll 725–738, Apr 2010.
     

  • [3] T. Bertrand et al., “Crystal structure of a four-copper laccase complexed with an arylamine: insights into substrate recognition and correlation with kinetics”, Biochemistry, vol 41, no 23, bll 7325–7333, Jun 2002.
     

  • [4] E. F. Pettersen et al., “UCSF Chimera--a visualization system for exploratory research and analysis”, J Comput Chem, vol 25, no 13, bll 1605–1612, Oct 2004.

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  • [5] Montagut Ferizzola, Yeison Javier, García Narbon, José Vicente, Jiménez Jiménez, Yolanda, March Iborra, Carmen, Montoya Baides, Angel, Torres Villa, Róbinson Alberto, Arnau Vives, Antonio , “Oscilador para biosensores basado en microbalanza de cristal de cuarzo (QCM)”, Diciembre 2011.

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  • [6]Fuchiwaki, Y., Tanaka, M., Makita, Y., & Ooie, T. (2014). “New approach to a practical quartz crystal microbalance sensor utilizing an inkjet printing system” October 2014

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  • [7] A. Joseph and A. Emadi, "A High Frequency Dual Inverted Mesa QCM Sensor Array With Concentric Electrodes," May 2020

References

In this way, the result is a functional quartz crystal microbalance, with a wide number of applications. This, together with the developed software, provides an excellent tool to perform mass measurements of the order of micrograms, which can be analyzed in a continuous flow (when the affinity between the biofilm and the analyte is high), a flow with a certain time of pause (to obtain that a greater quantity of the sample adheres), or in a manual way (with the technique of micropipetting).

 

Some of the perks that are in the construction of our biosensor are:

 

  1. We can take advantage of its capacity to change the resonance frequency according to any surface mass change on the resonator [5].

  2. They can be used as sensor devices without the need for labeling agents. As an online sensor, it works based on the fact that changes in oscillation frequency are proportional to changes in the mass on the electrode of the surface. [6]

  3. The QCM counts with a higher oscillation frequency that leads to a greater sensitivity.

 

On the other hand, there are some disadvantages from our QCM

 

  1. The QCM has a non-uniform mass sensitivity across the electrodes.

  2. They also count with a lower frequency of operation [7]