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Latest revision as of 20:13, 27 October 2020

Microcin PDI gene cluster

Contains all the elements necessary to constitutively express the entire Microcin PDI gene cluster to produce and secrete the antimicrobial peptide Microcin PDI: mcpM: coding for microcin PDI, mcpI: coding for the immunity protein, mcpA: coding for the activator protein, mcpD and mcpB coding for the secreting proteins. mcpM, mcpI, mcpA are under the control of the same promoter and the sequence is organized as follows: a promoter (BBa_J23111), an RBS (BBa_J61127), the CDS for McpM-His (BBa_K3409003), an RBS (BBa_J61130), the CDS for McpI (BBa_K3409004), an RBS (BBa_J61118), the CDS for McpA (BBa_K3409005) and a double forward terminator (BBa_B0015). mcpD and mcpB are under the control of another promoter and the sequence is organized as follows: a promoter (BBa_J23118), an RBS (BBa_J61118), the CDS for McpD, (BBa_K3409006), an RBS (BBa_J1109), the CDS for McpB (BBa_K3409007) and a double terminator (BBa_B0015).

Usage

In the last few years, as antimicrobial resistance is increasing, different strategies are emerging to find alternatives to antibiotics. Bacteriocins are antimicrobial peptides (AMP) naturally synthetized by ribosomes. They are produced by bacteria in order to survive in highly competitive polymicrobial environments and have a variable spectrum. They are considered as non harmful for human use, are easily degraded by proteolytic enzymes and can target specific pathogens. Microcins are a type of AMP whose secretion is not lethal to the producing cell (resistant genes), in the contrary to colicins which are lethal to the producing cell. Most bacteriocins act by causing lethal membrane damages to the target bacteria. Microcin PDI (Proximity Dependent Inhibition) is a class IIa microcin meaning it is plasmid-encoded and does not undergo post-translational modifications.

Biology

Microcin PDI which was first identified in 2009 and requires close physical proximity between the producer and susceptible strains. MccPDI has a narrow spectrum and can kill several strains of E. coli: multidrug resistant E. coli, enterotoxigenic E. coli expressing F5 (K99) and F4 (K88) fimbriae, enterohemorrhagic E. coli O157:H7 and O26, as well as commensal strains such as E. coli K12. It has also been shown that Shigella is sensitive to MccPDI. It recognizes specific Outer Membrane Protein F motifs on the target and leads to lethal membrane damage. The MccPDI gene cluster was initially identified in the strain E. coli 25 plasmid. It is a locus of approximately 4.8kb and is composed of 5 genes which are each necessary for the appropriate expression, activity and secretion of microcin PDI:

MccPDI gene cluster originally found in E. coli 25 is not constitutive and is controlled by the EnvZ/OmpR Two Component System (TCS). In fact, knockout mutants of the TCS did not show a PDI+ phenotype. EnvZ is an osmotic sensor which upregulates the phosphorylation of OmpR (a transcription factor) which binds to the promoter region upstream of McpM, McpI and McpA. It was shown that the production of MccPDI was higher in M9 medium compared to LB medium. Furthermore, mcpD and mcpB are under the influence of another transcriptional regulation.

For this construction, the expression of MccPDI is constitutive. It contains the native coding sequences of each one of the 5 genes and we replaced the rest with promoters, ribosome binding sites (RBS) and terminators contained in the iGEM Registry of Standard Biological Parts.

Characterization

Assembly of the MccPDI cluster and expression in E. coli DH5α

Several white colonies are obtained on plate N°1 and N°2 (Figure 1A) and no colonies are counted on the control (plate not present). Thus, the transformation seems to have worked. Regarding the verification of the ligation, colonies needed to be tested after having completed a miniprep procedure to obtain the plasmid. The extracted plasmids of the colonies have been cut with restriction enzymes (RE) other than the one used for the BioBrick Assembly method. Then, NdeI is used to linearize the plasmid 31. SapI is used to cut twice plasmid 31 to obtain 2 distinct bands at 10 kb and 3.3 kb as depicted on Well N°2 Figure 1C. For plasmid 39 we did not have any RE available in our inventory to linearize it. Thus, we only used BglI to cut twice plasmid 39 to obtain 2 distinct bands at 6.7 kb and 1.6 kb as depicted on Well N°1 Figure 1C. For colony 3 of plasmid 39: On lane 4, three bands can be observed: one around 6.7 kb and one around 1.6 kb and a last one unexpected around 3.5 kb. The two first bands correspond to the expected results. Nevertheless, we proceeded with the verification step for the colony 3 of plasmid 39.

Validating the production of Microcin PDI by E. coli DH5α

To validate the production of McpM, the protein needs to be extracted and purified from the cellular culture via Ni2+ chromatography column and further revelation via SDS-Page and staining technique. The purification is done on the clone with plasmid 39, which was selected after the previous experiment (Restriction enzyme validation Figure 1.B).

The McpM size is 8.2 kDa. Therefore, we expect that in the lane of the purified cell lysate (elutions) a band appears at the size of 8.2 kDa. In the first lane, this band should be more intense than lanes where Elution 2 and 3 have been loaded. We also expect to have few bands in the flow-through, corresponding to potential waste material. Regarding the lysate, the same bands as the flow through might appear, plus one at 8.2 kDa corresponding to McpM. Finally, in the lane of the Laemmli no bands should appear.

The chromatography was not 100% efficient as the elution bands still contain many proteins (even though the band are less intense than for the total lysate). The molecular weight marker did not migrate very well as the well was a little blocked by polymerized acrylamide towards the top. The 10kDa did not migrate correctly and is not horizontal. The bottom of the gel is not perfect, and it is difficult to visualize proteins. A light band can be observed at a weight inferior to 10kDa in the elution 2 lane. This band is expected to be the McpM which is 8.2kDa and which is not necessarily supposed to be present in large amount. However, we cannot be sure of the presence of McpM in well 3 (elution 2). To completely validate the presence of McpM in the purified cell lysate, a Western blot and immunodetection using an anti-His antibody should be performed. This is done by transferring the proteins from the gel to a nitrocellulose membrane and then performing the immunodetection, with a direct rabbit anti-His antibody conjugated to Horseradish Peroxidase, then detecting color using the HRP color detecting reagent. This will allow us to fully confirm that the low-intense band present <10kDa corresponds to McpM. For a matter of time and available equipment, we were not able to perform the Western Blot. Instead, we performed an agar diffusion assay to validate the production and activity of McpM (see below) by the clone containing plasmid 39.

Validating the secretion and antimicrobial activity of Microcin PDI by E. coli DH5α

We performed an agar diffusion assay to assess the correct antimicrobial activity of Microcin PDI produced by our chassis against the target.

The E. coli DH5 alpha (BB1,BB2,BB3) should kill the surrounding plated E. coli DH5 alpha when it expresses McpM. Therefore, there is a formation of radiuses around their colony (white 6-mm filter discs). This observation confirms the ability of the engineered cells to kill E. coli DH5 alpha bacteria using McpM. This antimicrobial activity is what we are aiming for phase 2 of the BacTail project.

Results: We tested in duplicate colony with plasmid 39 assembled with BioBrick 1,2, and 3 (Disc N°2 and N°4), in duplicate colony with plasmid 31 assembled with BioBrick 1,2, and 3 (Disc N°1 and 3), in duplicate E. coli DH10B strain containing the native MccpDI cluster (Disc N°5 and 7) (positive control) and as a negative control only LB medium (Disc 6).

To discuss the results, we zoomed on each disc of both assays, summarized in the table below. Two characteristics are taken into consideration during observation: presence or absence of a radius around the disc (due to the secretion of McppPDI microcin) and if some colonies have grown onto the disc or if the disc remains white.

Conclusion

Around the original strains (Disc 5 and 7 - positive controls) a small radius (1mm) is observed, and no red colonies have grown on the disc perimeters, which suggests that the E. coli DH5 alpha of the bacterial lawn have been killed or could not grow in this area of the plate due to the presence of the original MccPDI cluster. On the contrary, around the negative control (Dis 6 – LB medium only), no radius can be observed, and red colonies could grow on the disc, which shows that nothing hinders the E. coli DH5 alpha of the bacterial lawn to grow on this region. Regarding our samples, we performed the agar diffusion assay with the candidates kept after the construction validation (Figure 1B). We could observe on Disc N°2 Assay N°1 (the construction of plasmid 39), that no red colonies have grown on the disc perimeters and a small radius (1 mm) appeared on the disc perimeters. Additionally, a three-time bigger radius (3mm) on the disc N°2 Assay N°1 (construction of plasmid 39) could be observed. This difference in radius size can be explained by the higher volume that has been used for Disc N°2 Assay N°2. Finally, this observations shows that the construction of plasmid 39 has the same effect than the one of the original MccPDI cluster, meaning to induce the growth of the E. coli DH5 alpha of the bacterial lawn. This suggest that the construction of plasmid 39 expresses the McpM responsible for killing E. coli DH5 alpha.

Bibliography

Characterization of a novel microcin that kills enterohemorrhagic escherichia coli O157:H7 and O26. Applied and Environmental Microbiology, 78(18), 6592–6599. https://doi.org/10.1128/AEM.01067-12

Lu, S. Y., Graça, T., Avillan, J. J., Zhao, Z., & Call, D. R. (2019). Microcin PDI inhibits antibioticresistant strains of Escherichia coli and Shigella through a mechanism of membrane disruption and protection by homotrimer self-immunity. Applied and Environmental Microbiology, 85(11), 1–18. https://doi.org/10.1128/AEM.00371-19

Sawant, A. A., Casavant, N. C., Call, D. R., & Besser, T. E. (2011). Proximity-dependent inhibition in Escherichia coli isolates from cattle. Applied and Environmental Microbiology, 77(7), 2345–2351. https://doi.org/10.1128/AEM.03150-09

Simons, A., Alhanout, K., & Duval, R. E. (2020). Bacteriocins, Antimicrobial Peptides from Bacterial Origin: Overview of Their Biology and Their Impact against Multidrug-Resistant Bacteria. Microorganisms, 8(5), 639.

Zhao, Z., Orfe, L. H., Liu, J., Lu, S. Y., Besser, T. E., & Call, D. R. (2017). Microcin PDI regulation and proteolytic cleavage are unique among known microcins. Scientific Reports, 7(February), 1–14. https://doi.org/10.1038/srep42529

Sequence and Features