Target Vector

Background

The Target Vector (BBa_K215002) is designed as the construct that will tag afp for use in the IPP system.

The first key feature of the Target's design is the NheI restriction site. NheI is compatible with XbaI and SpeI, meaning that a biobrick digested at the X and S sites can be ligated into the Target Vector at the NheI site (for detailed protocol see here!!!). At the N-terminus of the Target is the display (aka Nano) tag, which is a 15 amino acid sequence that binds the target protein to streptavidin or some analogous protein. At the C-terminus of the Target is the 180 amino acid Secretion tag (aka prtB) that is recognized and secreted to the extracellular space by the IPP's secretion system. Flanking each side of the NheI site are His tags and TEV (protease) sites; these allow for both traditional IMAC purification of the target protein and cleavage of the tags from afp after it has been purified.

The display/Nano tag was retrieved from Lamla and Erdmann: [1]

The secretion tag/prt B was retrieved from Palacios et al: [2]

The TEV Recognition Site, ENHLYFQG, was retrieved from [3]

Experiments

We inserted and characterized two proteins within the Target Vector: GFP (BBa_E0040) and Opda (BBa_K215090). (For more background information on the enzyme Opda, see here).

After insertion of E0040 into the target construct, the tagged GFP (target-GFP) was found to still be soluble and fluorescent. The standard curve below shows how the fluorescence of the target-GFP correlates to its concentration. This curve was generated using target-GFP that was purified using a traditional IMAC technique.

After insertion of K215090 into the target construct, the tagged Opda (target-Opda) was found to be....

Insert target-Opda data here!!!