Team:Stockholm/Part Collection

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Proof of concept Nuc Lys Def Esp Sort A

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The Sortase Package is a generalized solution, designed to enable conjugation/immobilization of any protein of interest to a fusion partner with an exposed glycine residue.
The Sortase A package consists of three key parts:

Sortase A BioBrick: A BioBrick encoding for Sortase A.

Linker-tag (LT) sequence: A custom designed linker-tag sequence encoding for the Sortase A recognition motif, LPETGG, and a histidine tag for purification purposes (Figure 1).

Protein primer design solution: A primer designed to anneal downstream the protein coding sequence with a BamHI overhang.

Sortase A BioBrick - BBa_K2144008

This BioBrick encodes for a truncated version of the transpeptidase Sortase A, fused with the Protein G B1 domain (GB1) acting as a solubility tag.

Linker-Tag - BBa_K2144101

The linker-tag sequence has a BamHI restriction site upstream its sequence. Digesting the BamHI and standard suffix PstI site enables a ligation of the LT sequence with any protein of interest when used in combination with the “protein primer design solution” and the vector's suffix PstI site.

Figure 1: An illustration of the main features in the custom designed linker-tag sequence

Protein Primer Design Solution

Designing primers for use with our vector containing the LT sequence might be easier with a few pointers:

• The forward strand of your protein should be coding so that the C-terminus of your protein is fused to the linker-tags. Sortase A conjugates C-terminal LPXTG to N-terminal poly-G sequences.
• Make sure to avoid including the stop codons or terminators typically used at the C-terminal of coding constructs as they will interrupt translation of the LT downstream of your protein.
• The primers have to be carefully tested since some of them require the insertion of an extra base before the BamHI restriction site in order to avoid a frameshift between protein and LT.
• Include a few extra bases on the 5’ end – BamHI does not digest efficiently at the very end of DNA molecules.

A primer we designed for our lysostaphin BioBrick can be seen below.

Figure 2: An example for a primer used in our SMITe project

By generalizing our Sortase package, we have designed a system in which future iGEM teams can easily utilize Sortase A to conjugate/immobilize any type of fusion partners of interest.

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