Cloning lumbrokinase with flanking restriction enzymatic sites

To clone lumbrokinase (BBa_K4673017) from red earthworms, we design the pairs of cloning primers according to the cDNA sequence of lumbrokinase, since the earthworms belong to eukaryotes and the genes contain introns that need to be concerned in the following protein expression. As a consequence, we referred to the NCBI Nucleotide database and chose the accession of lumbrokinase “AY187629” to design primer pairs. Because we planned to subclone lumbrokinase to pET-22b (+) vector which contain N’ pelB signal peptide (BBa_J32006:Design) and C’ 6x His tag (BBa_K4673028), we thus excluded the start codon “ATG” and stop codon “TAA” in the lumbrokinase cDNA. In addition, to successfully express lumbrokinase inframe, we designed BamHI or XhoI restriction enzymatic sites in 5’ or 3’ respectively. All in all, the sequences of the primer pairs are shown below. Please note that the lower case letters indicate artificial sequence that is not present in lumbrokinase cDNA and the underlying letters refer to the restriction enzyme recognition sites.

BamHI_LK_AY187629_F cgcggatcccGAACTTCCTCCCGGAACAAAA
XhoI_LK_AY187629_R ccgctcgagcggGTTGTTGGTGATGATGTCGGT

Replacing LacO by pFadH

To achieve oleate induction, we designed a primer that would substitute the Lac operator (BBa_K4673024). The primer is composed of two sticky ends, T7 promoter, and Fad R regulator (BBa_K4673025), which had the forward and reverse sequence. We used sticky ends BglII and XbaI since we cut the Lumbrokinase_pET-22b with restriction enzymes BglII and XbaI, thus using sticky ends BglII and XbaI, our primer can be successfully inserted into Lumfrokinase_pET-22b. We used Fad R regulator because Fad R regulator is a regulator of fatty acid metabolism, it can serve as an induction trigger of the expression of lumbrokinase. We selected T7 promoter because it is a very efficient promoter, thus lumbrokinase expression can be strong when induced by fatty acid.

T7_FadR_forward:
gatcTAATACGACTCACTATAGGCGACGGCTAAATTAGAACTCATCCGACCACAT

T7_FadR_reverse:
ctagATGTGGTCGGATGAGTTCTAATTTAGCCGTCGCCTATAGTGAGTCGTATTA