Difference between revisions of "Part:BBa K1152013"

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IndC Indigoidine Synthetase device

This part encodes the non-ribosomal peptide synthetase that synthesizes the blue pigment indigoidine. The IndC gene was amplified from P. luminescens laumondii TT01 (DSM15139) and cloned into pSB1C3.

Usage and Biology

The single module NRPS indC from P. luminescens contains an internal oxidation domain (Ox-domain) in its A-domain and a special TE-domain . This enzyme first adenylates L-glutamine (A-domain), which is then attached to the T-domain. The TE-domain cleaves and catalyzes the cyclization of the substrate, which is further oxidized by the Ox-domain. The oxidation of two cyclic glutamines results in the formation of the insoluble blue pigment indigoidine.

Sequence and Features

Successful cloning of the IndC expression device was confirmed by restriction digested (Figure 1) followed by sequencing.

Figure 1: Restriction digest of BBa_K1152013 with EcoRI, XbaI, SpeI and PstI (construct marked with red circle) confirms correct cloning of the part into pSB1C3.

Expression of functional indigoidine synthetase indC derived from P. lumninescens in five substrains of E. coli

The open reading frame of the native indigoidine synthetase indC was amplified from genomic DNA of P. lumninescens and cloned into a plasmid under the control of an lac-inducible promoter. This indC expression cassette was transformed into different substrains of E. coli, namely DH5alpha, MG1655, BAP1, TOP10 and NEB Turbo. All of these host strains express the endogenous PPTAse entD which is responsible for the transfer of the 4'-phosphopantetheine residue from coenzyme A to the apo-domain of EntF, a T-domain in the enterobactin pathway(). As depicted in Figure 2a, entD does not exhibit strict substrate speceficity in being restricted to activating domains of the enterobactin pathway, but is able to activate the T-domain of indC as determined by the blue phenotype of the transformed cells. Except for NEB Turbo cells, all transformed host strains displayed a decelerated growth and significantly smaller colonies on plate when compared to the negative control. The blue phenotype developed late after transformation ranging from first blue colonies after 24 h and taking up to three days for visible poduction of the blue pigment. NEB Turbo showed regular colony growth and developed a strong blue phenotype upon induction with IPTG. As all host strains were able to express the functional indigoidine sythetase derived from a different species, further experiments were only conducted with one E. coli strain. Due to its simplicity in handling and sufficient expression of the constructs, the substrain TOP10 was chosen. Improved Production of Indigoidine by Co-transformation of Host Strain with Supplementary PPTases The expression of indC under activation by endC was sufficient for easy detection of indigoidine production on plates harboring indC-carrying cells. In order to determine, whether the amount of indigoidine production in the E. coliTOP10 cells is dependent on the quality of the interacting of indC with the PPTase, four PPTase dervied from varying origins were selected and amplified from the genome of the hosts of origin. E. coliTOP10 cells were co-transformed with plasmids coding for the different PPTases and the plasmid containing the expression cassette for indC. As reference for the endogenous PPTase activity served cells only transformed with the indC plasmid. Irrespective of the PPTase, growth of colonies was retarded. Remarkably however, colonies co-transformed with the PPTase plasmid remained of smaller size than the ones only carrying the indC construct. On the other side, indigoidine production was more diffuse in the latter cells with secretion of the blue pigment into the agar (Figure 2b, indC) and only slight blue-greenish coloring of the colonies. The four PPTases additionally introduced into the TOP10 cells were all shown to be functional (blue phenotype of the transformants, Figure 6b), but lead to the retention of most of the indigoidine within the cells. Colonies of cells transformed with thess constructs, were of convex shape and of distinct, dark blue color. Overall, cells carrying an additional PPTase showed increased indigoidine production compared to the cells relying on the endogenous entD.

We tested the IndC expression contruct by cotransformation in different E. coli strains and in combination with different PPTase expression constructs (Figure 2).

Figure 2: Comparison between different E. coli strains and PPTases: a) Comparison of different E. coli strains examining growth and indigoidine production The figure shows five different strains of E. coli that have been co-transformed with an indC expression plasmid and a sfp expression plasmid. The negative control is E. coli TOP10 without a plasmid. All transformants have been grown on LB agar for 48 hours at room temperature, cells were not induced. One can see that even without induction all strains express the indigoidine synthetase and produce the blue pigment indigoidine. However, the strains BAP1 and NEB Turbo grow faster in the first day, exhibiting a white phenotype (data not shown). Colonies on the plate of E. coli TOP10 are very small and dark blue/ black. Assuming that indigoidine production inhibits cell growth due to its toxicity, we concluded that TOP10 produced the most indigoidine among the strains we tested. We used E. coli TOP10 for the following experiments. b) Comparison between different PPTases concerning overall indigoidine production The Figure shows E. coli TOP10 cells co-transformed with indC and four different PPTases (sfp, svp, entD and delC), respectively. The image bottom left shows E. coli TOP10 cells without additional PPTase and the negative control is TOP10 without a plasmid.

Source

IndC was amplified from the P. luminescens laumondii TT01 (DSM15139) genome.

References