Grafting with allograft is well established, but there are problems with availability, cost, infection, antigenicity, reproducibility, and stability of the created construct.
In creating constructed novel ecosystems within the OCBIL landscapes of Western Australia, the techniques within a baroque toolbox succour the local by embracing and enhancing intrinsic ephemera and site specificities.
Synthetic biologists claimed that they could do even do more than 'just' writing; that they could create, construct, indeed, 'engineer' or 'design'artificialalifefe' (Cserer and Seiringer 2009; Hellsten and Nerlich 2011).
The PCR fragment was sub-cloned into pGEMTeasy vector (Promega), and ligated to pGL3-basic (SmaI site), creating construct 375a, or to TK-luc.
To determine if this system could be used to measure FRET between internal sites of RyR1, additional fusion proteins were created that contained a His10 tag at amino acid residue 1861 in divergent region 3 (DR3) [17] and GFP at either the N-terminus (to create construct GFP(1 DR3His) or at amino acid position 618 (to create construct GFP 618 DR3His (Fig. 7a)).
This cDNA was cloned behind a 35S cauliflower mosaic virus promoter in a T-DNA vector to create construct 35S::PCN-miRNAd, and transformed into Populus using an Agrobacterium-based method (Materials and Methods).
To allow subsequent 'repair' of any deletion, the LEE4 operon was amplified by long-range PCR and cloned into pIB307 to create construct pAJR162.
To create construct 35S+LI, the mature 5'UTR present in construct 35S was replaced with the 5'UTR of AtMHX that included the LI, which was amplified by PCR from the WT construct.
We created constructs encoding the full-length S1R with APEX2 attached at either the N- or C-terminus of the receptor (Fig. 1B).
To identify amino acids in SMN that are important for calpain cleavage, we created constructs containing internal deletions in EGFP-SMN and tested their susceptibility to calpain cleavage in cell-free assays.
