Part:BBa_K5033005

OncoBiotica: mFadA[B]_GSLinker_CDA[Theta]

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 'Theta' mutant of the basic part BBa_K5033000. Within the codA cytosine deaminase (CDA) domain, it contains a V152A (the valine on position 152 has been exchanged with alanine), a F316C (the phenylalanine on position 316 has been exchanged with cysteine)an a D317G (the aspartatic acid on position 317 has been exchanged with glycine) mutation. In the context of our fusionprotein they are V190A, F354C and D355G mutations. This mutant has been selected for research in the lab with the help of literature research and modeling.
Its main use is to understand the calalytic activity of our fusionprotein.
It is a variant 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 showed that .... 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), 'Beta' (BBa_K5033002), 'Gamma' (BBa_K5033003) and 'Epsilon' (BBa_K5033004).

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 V152A, F316C and D317G mutations of the enzyme.
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

V152A/F316C/D317G has been previously reported to exhibit improved efficiency and specificity compared to the WT and the D314A mutation. Specifically its relative efficiency and specificity towards 5-FC are 1.53 and 20.5 times higher than the WT.[1] According to CompassR [2] predictions, the triple mutant also displays increased stability compared to the WT, though not as high as D314A (ΔΔG_fold = -1.05 kcal/mol). We sought to investigate the structural changes induced by this triple substitution.

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[Theta] as described by Benchling.

Protein Structure Prediction

Figure 3: Tertiary structure of mFadA[B]_GSLinker_CDA[Epsilon] 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.

In our generated model with 5-FC as the ligand, all substituted amino acids are not present in the active center but appear to be in its direct vicinity. The important amino acids (E217, H246, and D313) for the reaction are all predominantly in the correct conformation relative to the iron and substrate. Both A152 and C316 appear to have no direct influence on the structure of the active center. However, G317 shifts away the negatively charged carboxyl group of D314 from the fluorine atom (from 2.801 Å to 3.060 Å). This reduces the electronegative charge, thereby creating a more stable environemt for the strongly electronegative fluorine. This effect parallels that of the D314A mutation, where the replacement of a carboxyl group with a methyl group similarly improved the environment for fluorine.[4] In contrast, the model with cytosine as ligand reveals significant alterations in the active center. As observed in the R91T/D314A mutant, the pyrimidine ring of cytosine no longer aligns parallel to W319 but instead faces towards it. This reorientation places the amine group on the opposite site of D313 preventing any interaction between D313 and the amine group of cytosine. Given that D313 is essential for the mechanism that exchanges the amine group with oxygen, this likely results in reduced affinity for cytosine. Consequently, the observed conformation may shift the enzyme's selectivity towards 5-FC. These findings are consistent with the data presented in the original study that characterized this mutant.[1]

Cloning of the Plasmid

To build the plasmid containing the gene for our Theta 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 Theta 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[Theta] 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[Theta] 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 6: SDS pages 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 51.99kDA (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.

UV-Vis

An assay using a spectrometer was devised. This measures the peak of the absorbance spectrum, which shifts to lower wavelengths as the ratio of 5-FU to 5-FC increases. We established a linear relationship, which we calibrated using standards. More details can be found in the UV-Vis Peak Shift Assay protocol on our Experiments page.
Note that this method does not give a high resolution beyond 5% 5-FU/5-FC intervals.

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.

Data and conclusion

Sadly the measurements of the Theta variant with HPLC did not provide valuable data within our projects scope. If there would have been more time we would redo the assays to obtain better chromatograms, that can be analyzed.

Using Uv-Vis assays we were able to show that this variant is fast in comparison to the wild type fusionprotein. Since the mutations this variant is based on, are already described in literature to be promising, we sort of reproduced the litureture with our approach.

Our novel muation combinations Beta (BBa_K5033002), Gamma (BBa_K5033003) and Epsilon (BBa_K5033004) were more interesting to us. Using modeling, HPLC and ¹⁹F NMR (for Gamma and the wild type), we have shown that the Gamma variant is the most promising novel variant.

References

[1] Fuchita, M., Ardiani, A., Zhao, L., Serve, K., Stoddard, B., Black, M., 2009. Cancer Res 69 (11): 4791–4799.. https://doi.org/10.1158/0008-5472.CAN-09-0615
[2] Cui, H., Cao, H., Cai, H., Jaeger, K., Davari, M.D., Schwaneberg, U., 2020. Computer‐Assisted Recombination (CompassR) Teaches us How to Recombine Beneficial Substitutions from Directed Evolution Campaigns. Chemistry – A European Journal 26, 643–649.. https://doi.org/10.1002/chem.201903994

[3] PDB Entry - 1RA0. https://doi.org/10.2210/pdb1RA0/pdb
[4] modeling

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]