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1. Sensitivity test : Results

     Fig 1: Functional test for plasmid carrying the MccB17 operon (pCID909). Supernatant of cultures of MC1061 without any plasmid (MC1061), or containing a plasmid carrying the microcinB17 operon (McB17-3 and McB17-4) or containing a plasmid carrying the Mcc7 operon (Mcc7) were spotted on a lawn of MC1061 strain containing a plasmid carrying the MccB17 operon.

     MC1061 producing Microcin B17 (lawn) is sensitive to Microcin C7 as a growth inhibition halo around the supernatant drop is observed. On the other hand, this strain is immune against Microcin B17 (clones 3 and 4). As a control, MC1061 supernatant did not cause any growth inhibition. Thus, a strain producing Microcin B17 is immune against Microcin B17 as expected since the operon encodes the immunity gene but remains sensitive to Microcin C7. In conclusion, there is no cross-immunity i.e. the immunity is highly specific.

     Fig 2: Functional test for plasmid carrying the MccC7 operon (pp70). Supernatant of cultures of MC1061 without any plasmid (MC1061), or containing a plasmid carrying the microcinB17 operon (McB17-3 and McB17-4) or containing a plasmid carrying the Mcc7 operon (Mcc7) were spotted on a lawn of MC1061 strain containing a plasmid carrying the MccC7 operon.

     MC1061 producing Microcin C7 (lawn) is sensitive to Microcin B17 (clones 3 and 4) as a growth inhibition halo around the supernatant drop is observed. On the other hand, this strain is immune against Microcin C7. As a control, MC1061 supernatant did not cause any growth inhibition. Thus, as observed for Microcin B17, immunity against Microcin C7 is highly specific and no cross-immunity is observed.

     Microcin C7 bacteria seem then to be resistant to their own microcin production but sensitive to microcin B17. Sensitive strain’s supernatant had no impact on bacterial growth.

      Fig 3: Functional test for plasmid carrying the MccC7 operon (pp70) or the MccB17 operon (PCID909). Supernatant of cultures of MC1061 without any plasmid (MC1061), or containing a plasmid carrying the microcinB17 operon (McB17-3 and McB17-4) or containing a plasmid carrying the Mcc7 operon (Mcc7) were spotted on a lawn of MC1061 strain.

     MC1061 (lawn) is sensitive to Microcin C7 as a growth inhibition halo around the supernatant drop is observed. For unknown reason, the growth inhibition caused by Microcin B17 (clones 3 and 4) is weaker that what we observed for Microcin C7. As a control, MC1061 supernatant did not cause any growth inhibition.

     Thus, these experiments validated the basis of our model (see modeling): there is no-cross immunity between Microcin B17 and Microcin C7. Therefore, we might expect some selection in our competition experiments.

2. Construction of biobricks encoding the genes of Microcin B17 and C7

     In order to build the integron tool for pathway rearrangement, we achieved the construction of 7 out of 13 biobricks needed.

     To construct these biobricks, each genes of the microcin operons were amplified by PCR using specific primers. The upstream primers contain the RFC10 prefix sequence as well as a RBS. The downstream primers contain an attc site followed by a RFC10 suffix.

     The RFC10 prefix and suffix will allow inserting the microcin genes in iGEM plasmids and in the “integron” (pSWlib) plasmid of D. Mazel. This plasmid is an iGEM plasmid that contains the RFC10 site downstream of an atti site and the pL1 promoter. The attc site is essential to allow the rearrangement of the genes of the operon within the integron platform.

The sequence of the primers is available under the materiel and method tab.

     Fig. 5: PCR amplification on colonies containing pSBIK3 plasmid having incorporated the MccCF gene. PCR amplification were performed as described in Materials and Methods using the primers specific to MccCF. NC: negative control, SL: smart ladder, PC: positive control and CF: amplification of MccCF in pSBIK3.

     Fig 6: PCR amplification on colonies containing pSBIK3 plasmid having incorporated the MccBB, MccBC and MccC genes. PCR amplifications were performed as described in Materials and Methods using the primers specific to each gene. SL: smart ladder, BB3: clone 3 of MccBB, BC3: clone 3 of MccBC, CC10: clone 10 of MccCC and NC: negative control.

Conclusion:

     Each microcin producing bacteria strain is immun against to its own microcin production: microcin B17 expressing bacteria against microcin B17 and microcin C7 expressing bacteria against microcin C7. Bacteria that do not express any microcin operon are sensitive to both microcin but microcin C7 seems more toxic than B17.

     This last observation follows theory: microcin B17 causes cessation of DNA replication and SOS response activation by inhibiting DNA gyrase while microcine C7 inhibits the translation by targeting the synthesis of aminoacylated tRNA^Asp.