Part:BBa_K3409013
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
NOTE: In this section: BB1 = part BBa_K3409010 - BB2 = part BBa_K3409011 – BB3 = part BBa_K3409012
MCPM PRODUCTION VALIDATION
Extraction, purification and validation of McpM production
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.
Expected results
The McpM size is 8 kDa. Therefore, we expect that the in the lane of the elutions a band appear at the size of 8 kDA. In the first lane, this band should be dark and be less intense in the lane 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 can appear, plus one at 8 kDa. Finally, in the lane of the Laemmli no bands should appear.
MCPM SECRETION AND ACTIVITY VALIDATION
AGAR DIFFUSION ASSAY
Expected results
The E.coli DH5 alpha (containing this part) when it is expressing the McpM should kill the surrounding plated E.coli DH5 alpha and therefore, create a little radius around the white 6-mm filter discs. This observation would confirm their ability to kill E.coli DH5 alpha bacteria, the activity we are aiming for the phase 2 of the BacTail project.
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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 1498
Illegal NheI site found at 1521 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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