Part:BBa_K4370005:Experience

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Applications of BBa_K4370005

To characterize the PRK activity of Streptomyces bottropensis ATCC 25435 (BBa_K4370004), we analyzed the impact of its overexpression in E. coli chassis. Indeed, Parikh et al. (Parikh et al., Protein Eng Des Sel, 2006) previously reported that the overexpression of the PRK from Synechococcus PCC7942 is toxic in this chassis when arabinose is used as a carbon source.

A codon-optimized version of the prk gene of S. bottropensis ATCC 25435 (BBa_K4370004, with an RBS in BBa_K4370005) was cloned under the control of an RBS (BBa_K4370005) into the L-arabinose-inducible expression vector pBAD33. This plasmid or an empty pBAD33 vector (‘pBAD33-Ø‘) were introduced in two E. coli K12 genetic backgrounds, one being able to use arabinose as a carbon source (DH5α), the other not (BW25113). Bacteria were grown on M9 minimal (Sambrook and Russell, 2001) agar plates (supplemented with 2 mM MgCl₂, 0.1 mM CaCl₂ and 0.2 % thiamine) including 0.2 % glucose and/or arabinose and 30 µg/ml chloramphenicol.

After two days of growth, we observed that the induction of the expression of BBa_K4370005 is toxic to E. coli, only when the cells use arabinose as only carbon source. This confirms that the PRK from S. bottropensis is active, and impacts E. coli metabolism the same way the PRK from Synechococcus PCC7942 does.

In the course of this work, we designed a prk sequence from optimized from an expression in E. coli (BBa_K4370004, with an RBS in BBa_K4370005). After constructing and testing the vectors, we learned that the expression of the biobrick is toxic to these cells when arabinose is used as a source of carbon, as previously reported for another PRK. We learnt from this experiment that the genomic island present in S. bottropensis ATCC 25435 encodes a functional PRK enzyme.

Figure : Growth of E. coli>/i> strains harboring pBAD33-BBa_K4370005 or a control vector on M9 supplemented minimal medium in the presence of glucose and/or arabinose. Five µl of bacteria resuspended in M9 medium devoid of carbon source were spotted on the plate. The first dilution (‘10^-1’) was adjusted to OD_{600 nm} 0.4 for all strains. The experiments were performed independently for <i>ara⁺ (DH5) and ara^- (BW25113) strains.

References Monal R. Parikh, Dina N. Greene, Kristen K. Woods, and Ichiro Matsumura, “Directed evolution of RuBisCO hypermorphs through genetic selection in engineered E.coli”, Protein Engineering, Design and Selection 2006 Mar; 19(3): 113–119 https://doi-org.insb.bib.cnrs.fr/10.1093/protein/gzj010

Sambrook, J.; Russell, DW. Molecular Cloning: A Laboratory Manual. 3. Cold Spring Harbor Laboratory Press; Cold Spring Harbor, NY: 2001.

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