Part:BBa_K1973014

Pm (2)

Pm2 is one variant of the promoter for the xylS/Pm expression system. It was obtained by site-directed mutagenesis PCR from the Pseudomonas putida plasmid pMPO52 in order to remove illegal restriction sites. The expression of this promoter is activated by XylS or XylS2 in the presence of benzoate and salicylate, respectively.

We created the Pm promoter by mutagenic PCR,as described in Protocols (Site-directed mutagenesis using overlap extension PCR), so we do not know the effect of this mutation in a promoter and whether it could be converted to a constitutive or inducible by other molecule promoter, rise or reduce its basal or induce expression...

To test the correct function of the Pm promoters, we cloned the three variants of Pm (original Pm, Pm1, Pm2 and Pm3) with the fusion protein lacZ-gfpmut3 by triparental mating, as described in our wiki page for Protocols (Triparental mating), in the strains KT2442-TpMRB170 (nahR-Psal-xylS2) and KT2442-TpMRB159 (nahR-Psal-nasF-xylS2) that were generated by electroporation and transposition, as described in Protocols (Electroporation and transposition). We test the Pm promoters in LB and LB+salicylate 2 mM and our control is the plasmid with the fusion protein lacZ-gfpmut3 without promoter (pMRB1).

We test the constructs using the beta-galactosidase activity assay in 2 different conditions (LB and LB+salicylate 2 mM). We did 4 replicates of the strains with the transposon nahR-Psal-xylS2 (BBa_K1973024) and 2 replicates of the strains with the transposon nahR-Psal-nasF-xylS2 (BBa_K1973021). There is a significant difference between the expression of the promoters when the regulation module (expression of xylS2) is induced and when it is not induced. Furthermore, the strains with nahR-Psal-xylS2 have more expression when it is induced and when it is not induced in comparison with the strains with the attenuator.

The three variants of the Pm promoter work satisfactory and the Pm1 promoter (BBa_K1973013) is the variant that has the best expression level in the system without the attenuator when it is induced. On the other hand, the Pm3 promoter (BBa_K1973915) is the variant that has the best expression level in the system with the attenuator when it is induced.

Figure 1. Beta-galactosidase assay of Pm promoters. The image represents the beta-galactosidase activity of the different Pm promoters in the strains expressing xylS2 without nasF (A) and with the attenuator (B). We assay the strains KT2442-TpMRB170/pMRB1 and KT2442-TpMRB159/pMRB1 concurrently to compare the induction effect of the expression promoter.

For further information, check our wiki page http://2016.igem.org/Team:UPO-Sevilla/Experiments .

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 326
  
• 1000
  COMPATIBLE WITH RFC[1000]