4.4. Generation of the plasmids

To complement antJ in the TT01−1°ΔantJ deletion strain and for overexpression of antJ in the wild type, the entire coding sequence of antJ was amplified from genomic DNA of P. luminescens using primers AKHp14 and AKHp15. Using these primers on both ends of the insert a 24 bp overhang homologous region to pCOLA_tacI/I or pACYC_tacI/I respectively, was added to enable Gibson assembly. The backbones of pCOLA_tacI/I and pACYC_tacI/I were amplified by PCR using primers AKHp12 and AKHp13. Assembled products (pAKH04 and pAKH05) were used to transform E. coli DH10B. Plasmids were isolated and transferred into strains TT01−1°ΔantJ and TT01−1° or TT01−2°, respectively.

For the purification of AntJ used for SPR spectroscopy, the plasmid pBAD24-antJ-His was generated via amplification of antJ (728 bp) using primers antJ-NheI_fwd and antJ-NdeI-ostop_rev. The insert and the vector were cut with restriction enzymes NheI and NdeI. The correct insertion was verified via PCR using primers pBAD24-seq_fwd and pBAD24-seq_rev.

For the purification of AntJ used in EMSA assays the plasmid pCATI4_antJ was constructed. For that purpose antJ was amplified with the primers AKHp44/45 and to both ends of the insert an overhang homologous to pCATI4 was attached for Gibson assembly. The vector pCATI4 was amplified by PCR using primers AKHp42/43. E. coli DH10B was transformed with the assembled vector. The vector insert was verified via sequencing.

For reporter assays in E. coli LMG194, the plasmid pBAD24-antJ was generated via amplification of antJ with the primers antJ-NheI_fwd and antJ-NdeI_rev (731 bp) and subsequent cleavage of the insert and the vector via NheI and NdeI. The different constructs of the antA promoter region were cloned into the plasmid pBBR1-mcs5-tt-lux. The complete promoter was amplified with the primers PantA-XbaI_fwd and PantA-XmaI_rev (389 bp). For generation of the antA promoter derivatives s1 (241 bp), s2 (239 bp) and s3 (203 bp), the primers PantA-s1-XbaI_fwd, PantA-s2-XbaI_fwd and PantA-s3-XbaI_fwd were used and each primer was combined with PantA-XmaI_rev for PCR. The construct mm1 (398 bp PCR) was generated via an overlap-PCR (PantA-XbaI_fwd, PantA-XmAI_rev) of the products that had been amplified with the primers pairs PantA-XbaI_fwd, PantA-mm1-ol_rev and PantA-mm1-ol_fwd and PantA-XmaI_rev. The construct mm2 (398 bp) was made via an overlap-PCR (PantA-XbaI_fwd, PantA-XmAI_rev) of the products that had been amplified with the primer pairs PantA-XbaI_fwd, PantA-mm2_rev and PantA-mm2_fwd, PantA-XmaI_rev. For construction of the mspacer (398 bp) two PCRs with the primer pairs PantA-XbaI_fwd, PantA-spacer-ol_rev and PantA-spacer-ol_fwd, PantA-XbaI_rev and a subsequent overlap-PCR as described above were performed. All of the PCR products were cut with restriction enzymes XbaI and XmaI and subsequently ligated with equally treated vector pBBR1-mcs5-tt-lux. Correct insertions were verified via control-PCR using the primers check-pBBR_fwd and check-pBBR_rev.

A PCR was performed to amplify eyfp with a size of 757 bp by using the primers eyfp-mcs_fwd and eyfp-EcoRI_rev and the plasmid pEYFP as a template. The PCR product and the vector pPINT-mCherry [25] were cut with the enzymes ApaI and EcoRI and the resulting plasmid was called pPEINT-mCherry. The control-PCR was performed using the primers check-mcherry-ins_rev and check-eyfp2_rev with a resulting PCR size of 1106 bp.

In order to generate the eyfp fusions three different constructs were inserted into pPEINT-P_less-mCherry-P_less-eyfp. P_antJ (519 bp) was generated via PCR using primers PantJ-NheI_fwd and PantJ-Not_rev and for generation of P_antJ-antJ (1220 bp) primers PantJ-NheI_fwd and antJ-ostop-NcoI_rev were used. For generation of both PCR products chromosomal DNA of P. luminescens was used as template.

P_tac-antJ was amplified via Ptac-antJ-NheI_fwd and antJ-ostop-NcoI_rev, containing the lacI repressor gene and antJ under the control of the tac-promoter. The plasmid pAKH05 was used as template and the product size was 2110 bp.

A PCR was performed to generate P_antA-mCherry fusions for all three plasmids. P_antA was amplified by a PCR with the primers PantA-NheI_fwd and PantA-NheI_rev and the PCR product was cut with NheI. The proper insertion within the correct direction was verified via DNA sequencing. The resulting plasmids were called pPEINT-P_antA-mCherry-P_antJ-eyfp, pPEINT-P_antA-mCherry-P_antJ-antJ-eyfp and pPEINT-P_antA-mCherry-P_tac-antJ-eyfp. A control PCR with the primers check-mcherry-ins_rev and check-eyfp _rev was performed.

For generation of the plasmids pPINT-P_antJ-mCherry and pPINT-P_antJ-antJ-mCherry the following procedures were performed. P_antJ was amplified via the primers PantJ-NheI_fwd and PantJ-BamHI_rev resulting in a 516 bp product and the 1215 bp long P_antJ-antJ product was generated with the primers PantJ-NheI_fwd and antJ-ostop-BamHI_rev. For both PCR products, chromosomal DNA of P. luminescens was used as template. In order to insert P_antJ and P_antJ-antJ upstream of mCherry, the vector pPINT-mCherry as well as the inserts were cut with NheI and BamHI. The resulting plasmids were called pPINT-P_antJ-mCherry and pPINT-P_antJ-antJ-mCherry. The correct insertions were verified by a PCR with the primers check-mcherry-ins_rev and check-eyfp-ins_rev. Furthermore, all generated plasmids were sequenced (Genomics core facility, LMU Biozentrum) to verify correctness.