BioBrickTM compatible vector system for protein expression in Rhodobacter sphaeroides

Abstract

We report here the creation of a modular, plasmid-based protein expression system utilizing elements of the native Rhodobacter puf promoter in a BioBrick(TM)-based vector system with DsRed encoding a red fluorescent reporter protein. A suite of truncations of the puf promoter were made to assess the influence of different portions of this promoter on expression of heterologous proteins. The 3' end of puf was found to be particularly important for increasing expression, with transformants accumulating significant quantities of DsRed under both aerobic and anaerobic growth conditions. Expression levels of this reporter protein in Rhodobacter sphaeroides were comparable to those achieved in Escherichia coli using the strong, constitutive P lac promoter, thus demonstrating the robustness of the engineered system. Furthermore, we demonstrate the ability to tune the designed expression system by modulating cellular DsRed levels based upon the promoter segment utilized and oxygenation conditions. Last, we show that the new expression system is able to drive expression of a membrane protein, proteorhodopsin, and that membrane purifications from R. sphaeroides yielded significant quantities of proteorhodopsin. This toolset lays the groundwork for the engineering of multi-step pathways, including recalcitrant membrane proteins, in R. sphaeroides.

Three classes of oxygen-dependent cyclase involved in chlorophyll and bacteriochlorophyll biosynthesis

“…4B). To ensure that this result was due to deletion of rsp_6110, rather than a polar effect on acsF, rsp_6110 was expressed in trans in ΔbchEΔccoPΔ6110 from pBBRBB-Ppuf 843-1200 (27). Pigments from this strain were analyzed as above, and Mg-BChl a and Zn-BChl a were detected in this extract (Fig.…”

“…The pBB[gene] plasmid was used to express foreign genes in Rba. sphaeroides and was constructed by cloning the relevant ORF between BglII/NotI sites in pBBRBB-Ppuf 843-1200 (27). Rvi.…”

PufQ regulates porphyrin flux at the haem/bacteriochlorophyll branchpoint of tetrapyrrole biosynthesis via interactions with ferrochelatase

“…Δ pufQ mutant cells are orange in colour, as are many others harbouring mutations that abolish BChl biosynthesis. The pufQ gene was reintroduced to the Δ pufQ strain using the pBBRBB‐ Ppuf 843–1200 plasmid (Tikh et al ., ). pBBRBB‐ pufQ transconjugants displayed a WT‐like phenotype, with elevated levels of LH2 complexes (800 and 850 nm absorption), indicating that BChl biosynthesis has been restored and implying that the Δ pufQ phenotype does not arise from polar effects (Fig.…”

Augmenting light coverage for photosynthesis through YFP-enhanced charge separation at the Rhodobacter sphaeroides reaction centre

“…However, the absence of carotenoids in the Δ crtB strain used for this work could have created a need for photoprotective pigments, and it has been suggested that fluorescent proteins can play a photoprotective role2627. To confirm that the superior growth rate of the Δ crtB RC/YFP–LH1 strain (versus the Δ crtB negative control) was due to YFP energy transfer and not photoprotection (which could favour an increased growth rate), the expression plasmid pBBRBB–YFP, based on pBBRBB- Ppuf 843–1200 28, was used to make cytoplasmic YFP in Rba. sphaeroides Δ crtB , which is free of any attachment to membrane proteins.…”