pTrc99A vector

Marine sediment enrichment was used to construct a metagenomic library to recover tellurium from tellurite(a tellurium oxyanion)dissolved in water,followed by functional screening to discover new genes related to tellurite reduction.Transmission electron microscopy(TEM)reveals the formation of intracellular Te crystals in Escherichia coli cells transformed with specific DNA fragments from marine sediment metagenomes.The metagenomic fragment consisted of 691 bp and showed low homology to known proteins.Phylogenetic analysis showed that this metagenomic fragment is related to Pseudomonas stutzeri.Cloning and expression of the open reading frame(ORF)on the metagenomic fragment validated the role of this fragment in conferring tellurite resistance and tellurite-reducing activity on E.coli host cells.

To study functional ORFs in metagenomic fragments,two overlapping ORFs were cloned separately into the pTrc99A expression vector(purchased from Creative Biogene).Vector constructs used to express ORF1 and ORF2 are shown in Figure 1.The tellurite-reducing activity of the ORF1-expressing clone was verified by spotting assay,whereas the ORF2-expressing clone showed no tellurite-reducing activity(Figure 2).E.coli cells containing naive and empty vectors showed no significant growth or tellurite-reducing activity in spotting assays.Taken together,the results indicate that ORF1 is responsible for the tellurite reducing activity of E.coli strain A1.

Figure 1.Vector construct for expression of ORF1 and ORF2 using pTrc99A.(Munar M P,et al.2019)

Figure 2.Spot-plating was used to observe the tellurite-reducing activity of a E.coli strain A1 and b the ORF-expression clones on LB agar plates with 20μg/mL chloramphenicol(CHL)or 100μg/mL ampicillin(AMP)containing 1 mM Na₂TeO₃.WT:wild-type E.coli cells,WT/P1:wild-type E.coli carrying empty vector pHSG398,WT/P2:wild-type E.coli carrying empty vector pTrc99A,A1:E.coli strain A1;A1-ORF1 and A1-ORF2:recombinant E.coli clones containing pTrc99AORF1 and pTrc99A-ORF2 respectively.(Munar M P,et al.2019)