Part:BBa_K5033002

OncoBiotica: mFadA[B]_GSLinker_CDA[Beta]

If you are interested in an overview of the parts designed by the iGEM Team Aachen 2024, visit our Parts page.

This part, developed by iGEM Aachen 2024, is our 'Beta' mutant of the basic part BBa_K5033000. Within the codA cytosine deaminase (CDA) domain, it contains a F186W (the phenylalanine on position 186 has been exchanged with tryptophane) as well as a D314A mutation (the aspartic acid on position 314 has been exchanged with alanine). In the context of our fusionprotein they are F224W and D352A mutations. This mutant has been selected for research in the lab with the help of literature research and modeling.
Its main use is to see if we can improve the catalytic activity of our fusionprotein.
It is an optimized version of BBa_K5033000 which served as the foundation for exploring the concept of microbiota-directed cancer therapy. This part encodes a fusion protein designed to combine two functionalities. Binding specific bacteria and having an optimized enzymatic function. This part is to be cloned into a vector based on an inducable expression system. iGEM Aachen 2024 used a pET21b(+) vector.
iGEM Aachen 2024 successfully demonstrated that the enzymatic function is enhanced in comparison to the CDA-wild type fusionprotein. This is one of five mutants analyzed by iGEM Aachen 2024 in addition to the CDA-wild type fusionprotein.
See the other four variants: 'Alpha' (BBa_K5033001), 'Gamma' (BBa_K5033003), 'Epsilon' (BBa_K5033004) and 'Theta' (BBa_K5033005).

Figure 1: Schematic view of the fusion protein's coding sequence.

Part Composition

The first protein domain is derived from the part BBa_K4990002 but has been codon optimized for expression in E. coli. It is the mFadA B-domain, found in various Fusobacterium strains. This part has already been well described by the iGEM23_CPU-CHINA team. This domain should be able to bind to FadA pili on Fusobacterium nucleatum and its former subspecies Fusobacterium nucleatum, F. polymorphum, F. vincentii, F. animalis via self assembly.
To further investigate the binding domain's functionality, iGEM Aachen 2024 created the basic part BBa_K5033006. This variant replaces the enzyme in our fusion protein with eGFP as a reporter protein.
The second functional protein domain is linked to the mFadA B-domain by a synthetic flexible linker consisting of Glycin and Serine in alternating order. This linker is eleven amino acids long.

This second functional domain of the fusionprotein is a mutant of the codA cytosine deaminase (CDA) that is native to E. coli. This mutant contains the F186W and D314A mutations of the enzyme. This Enzyme converts cytosine to uracil in its host organism but it is also able to convert 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU).[1]

In this case the Enzyme can be used for an enzyme directed prodrug therapy. To be precise, 5-FC is the non-toxic substrate and 5-FU is the active chemotherapeutic agent.
The fusionprotein encoded by this part also contains a downstream hexa-histidine tag for protein purification.

Protein Modeling

To find interesting mutations that shall be investigated in the lab, our team used a research based modeling approach.
Before transformation of this biological part (cloned into the pET21b(+) plasmid backbone), the structure of the expected fusionprotein was modeled.

Selection of the Mutant

Modeling of mutation recombinations using computer assisted recombination (CompassR)[2], 'Beta' (F186W/D314A) was the mutant with the highest stability among mutants with recombined mutations (ΔΔG_fold = -5.95 kcal/mol). It harbours a combination of the D314A substitution (as in 'Alpha' (BBa_K5033001)) combined with F186W, a published substitution which was proposed by CompassR to be slightly less stable than the wild type fusionprotein (WT) (ΔΔG_fold = 0.81 kcal/mol). This might be compensated by the stabilizing effect of D314A.

Biochemical Properties

The fundamental biochemical properties like molecular mass and extinction coefficient are important for a lot of synBio work done with proteins. To see an overview of these properties, have a look at figure 2.

Figure 2: Biochemical properties of mFadA[B]_GSLinker_CDA[Beta] as described by Benchling.

Protein Structure Prediction

Figure 3: Tertiary structure of mFadA[B]_GSLinker_CDA[Beta] as predicted by AlphaFold2. From left to right: mFadA[B] as an alpha helix (blue), the flexible linker, the enzyme-mutant and the freely accessible His-Tag (orange).

The tertiary structure has been predicted using AlphaFold2 by DeepMind. In this case it is especially important, that the binding domain and the His-Tag are freely available.

Modeling of Substrate and Active Site Interaction

Why RoseTTAFold All-Atom?

Proteins rarely act alone. Although substantial progress in the prediction of protein structures has been made, modeling of proteins and their ligands still remains challenging. The development of RoseTTAFold All-Atom (RFAA) aims to tackle this issue by building a neural network that is trained to accurately model general biomolecules containing a wide range of nonprotein components. In contrast to other tools that only include sequence based modeling, RFAA incorporates a graphical representation that models non-protein molecules at the atomic level, capturing their chemical bonds and interactions. In combination with the training data set that also includes ligand-bound protein structures from the Protein Data Bank (pdb), it allows RFAA to predict protein structures, ions and non-protein ligands. Interestingly, during our project, DeepMind released a new AlphaFold version (v3) that includes selected ions and ligands. However, an earlier release would not have been advantageous for us, as 5-FC and cytosine are not among the selected ligands that AlphaFold3 includes. Nevertheless, this shows that the improvements made this year mark a significant step forward, paving the way for more refined and accurate modeling of proteins and ligands in the future.

We observed a good overall structural alignment of the wild type enzymes' crystal structure [3] to the RoseTTAFold All-Atom model. Upon closer inspection of the active site, we noticed small differences in torsion angles of the side chains which naturally led to slight differences in bond lengths between amino acids and the ligand. However, these differences are inherent to the modeling process and do not reflect significant deviations. Therefore, they do not compromise the reliability of our approach for predicting structural changes in the mutants. This allowed us to apply the approach to the generated mutants by CompassR.

Modeling Results

Figure 4: Model of interactions of the amino acids in the active site of the fusionprotein with 5-fluorocytosine. As predicted using RoseTTAFold All-Atom.

The substitution of aspartic acid with alanine at position 314 seems to have the same effect as the single substitution in the D314A mutant in introducing a stabilizing methyl group at the vicinity of the fluorine atom (distance: 3.625 Å). There appears to be no influence by the substitution of phenylalanine with tryptophan at position 186 in the active site. It is reported that the single sustitution of F186W resulted in a mutant with both higher specificity and efficiency for 5-FC relative to the WT as well as lower specificity and efficiency with cytosine as ligand [4]. However, according to CompassR, the substitution has a slight destabilizing effect that could be explained by tryptophan being bulkier than phenylalanine due to the additional inol group in its side chain. As stated, this substitution has no negative effects on the stabilizing interaction of A314 with 5-FC's fluorine atom. So we could not observe significant changes induced by F186W although it is reported to results in a higher efficiency and specificity for 5-FC. So we thought it could still be an interesting combination to test in the lab.

Cloning of the Plasmid

To build the plasmid containing the gene for our Beta variant. We used the plasmid we already had for our WT-Fusionprotein (BBa_K5033000; pET21b(+)_mFad[A]_GSLinker_CDA[WT]). The gene sequence for this part contains a BamHI restriction site between the linker and the enzyme. The backbone contains a XhoI restriction site at the end of the gene insert.
After modeling of the Beta variant we ordered the gene fragment, encoding this variant. We made sure to include the correct restriction sites.

The backbone was prepared using the BamHI and XhoI restriction enzymes. After digestion, the cut backbone was cleaned up using an agarose gel and a gel extraction kit. The same was done for the Insert.

After gel cleanup the cut backbone and insert were ligated using the T4 Ligase.

To enhance the efficiency of the plasmid transformation into E. coli BL21 (DE3) the plasmid was first propagated via transformation in E. coli DH5α.

The propagated pET21b(+)_mFadA[B]_GSLinker_CDA[Beta] plasmid could then be purified with a plasmid miniprep kit and used for transformation into the production organism E. coli BL21 (DE3).

Producing the Fusionprotein

After successful transformation of the pET21b(+)_mFadA[B]_GSLinker_CDA[Beta] plasmid into the production organism E. coli BL21 (DE3) the protein could be expressed and purified. The pET21b(+) backbone has a lac operon (including the lacI repressor), which can be induced with IPTG (IUPAC: Propan-2-yl 1-thio-β-D-galactopyranoside).

Expression and Purification of the Fusionprotein

The fusionprotein was expressed by adding IPTG to the medium to a final concentration of 1mM.
The His-tagged protein was purified using a Protino Ni-IDA 2000 packed column by Macherey & Nagel®.

Figure 5: SDS page showing the proteins in the elution fractions. The number corresponds to the imidazole concentration (in mM) in the elution buffer respectively. Example: E10 is an elution buffer with 10mM imidazole.

The fusionptrotein is expected to have a molecular weight of 52.11kDA (cf. Fig. 2). This corresponds to the big bands visible on the gel.
This gel shows that the E10 fraction still has a lot of impurities. The E50 fraction was desalted and stored in 50mM TRIS buffer, to use for the kinetic assays.

Kinetic Assays

If you are interested in the methods used, take a look at our Experiments page.

High-Performance Liquid Chromatographie (HPLC)

We used Reverse Phase High Performance Liquid Chromatography for quantitative Analysis of 5-fluorocytosine and 5-fluorouracil in mutual solution. The results seen below were all measured with the same method. (see Experiments page)
Standards at between 10 µM and 500 µM were made to translate the peak area into compound concentration. After measuring, the chromatograms were evaluated with “OpenChrom” by Lablicate. For this, a baseline subtraction filter was applied, after this the standard first derivitave peak detector and trapezoid peak integrator were run. We identified para-aminobenzoicacid as a potential internal standard, but no problems which would necessitate the use of an internal standard arose.

Figure 6: Example HPLC Chromatogram obtained from measurement using 400 µM 5-FC and 200 µM 5-FU; the first peak being the 5-FC, the second 5-FU.

Enzyme Kinetics

Similar to the wild type enzyme, the mutant cytosine deaminase followed Michaelis-Menten kinetics. However, due to the limited solubility of 5-fluorocytosine (5-FC), achieving substrate concentrations sufficient for determining the mutants' maximum velocity (V_max) was difficult, leading to variability in kinetic measurements. This made the direct determination of K_m and V_max unreliable. To assess the mutants' performance, three key metrics were employed: (1) the time required to reach half-maximal product formation t_1/2RP, (2) the initial reaction rate (initial velocity) v₀, and (3) the total product formed over time as a measure of relative efficiency RFA.

For each mutant and method (HPLC, NMR, and UV-Vis Spectroscopy), relative substrate and product concentrations were plotted against time, and these graphs formed the basis of the analysis. The time points at which half-maximal product formation was reached were graphically determined, and substrate and product concentrations for each variant are plotted against time in figure 7. As with the wild type, the relative efficiency of the mutant was calculated by determining the area under the graph within a predefined time interval, assuming linearity between data points. This area represents the total product formed over time and provides a valuable comparison of efficiency of the mutant.

Figure 7: Representation of the relative amount of substance n_rel for the Alpha mutant against the time t obtained from HPLC measurements.

The initial velocities were determined by linear approximation of relative product formation during the first 80 seconds for the HPLC method. Each mutant's performance was directly compared to the wild type by plotting against the time, with the slope of the regression line representing the relative velocity for each condition.

The relative activity of each mutant was calculated as the mean of the two key parameters: the time to half-maximal product formation and the initial velocity, with the standard deviation providing an estimate of error. This combined measure of relative activity, allowed for a direct comparison of each variant's performance (cf. fig. 8).

Figure 8: Summary of the obtained RPA, t_(1/2)RP and v₀ values from HPLC measurements.

Figure 9: Comparison of the relative enyzme performance for the wild type and Alpha, Beta and Gamma variants; data obtained from HPLC.

This detailed approach allowed us to quantify the relative activity and efficiency of each mutant, highlighting the differences in their performance compared to the wild type.

Conclusion

We were able to show, that the Beta variant an increased catalytic performance in comparison to the wild type fusionprotein (cf. fig. 9).

Figure 10 also shows, that beta has a lower relative activity than our most promising Gamma variant. However, in NMR, Gamma showed only 1.2 times the efficiency compared to the wild type, likely due to the suboptimal conditions of very low enzyme concentrations and extended reaction times.
When comparing Betas' and Gammas' HPLC data, Gamma is the clear winner.

In addition to this Gamma is expected to have a higher relative affinity to our substrate 5-FC, too see more on this, visit Gammas' Part page.

References

[1] Aučynaitė, A., Rutkienė, R., Tauraitė, D., Meškys, R., Urbonavičius, J., 2018. Discovery of Bacterial Deaminases That Convert 5-Fluoroisocytosine Into 5-Fluorouracil. Frontiers in Microbiology 9.. https://doi.org/10.3389/fmicb.2018.02375
[2] Sheri D. Mahan, Greg C. Ireton, Catherine Knoeber, Barry L. Stoddard, Margaret E. Black, Random mutagenesis and selection of Escherichia coli cytosine deaminase for cancer gene therapy, Protein Engineering, Design and Selection, Volume 17, Issue 8, August 2004, Pages 625–633, https://doi.org/10.1093/protein/gzh074
[3] PDB Entry - 1RA0. https://doi.org/10.2210/pdb1RA0/pdb
[4] Kohila, V., Jaiswalb, A., Ghosh, S., 2012. Rationally designed Escherichia colicytosine deaminase mutants with improved specificity towards the prodrug 5-fluorocytosine for potential gene therapy applications. Med. Chem. Commun., 2012,3, 1316-1322 https://doi.org/10.1039/C2MD20209C

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 115
  Illegal XhoI site found at 1396
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1254
  Illegal NgoMIV site found at 1341
  
• 1000
  COMPATIBLE WITH RFC[1000]