Sortase Cloning

 

Post Type: protein_labelling
As will be discussed
http://blog.openwetware.org/scienceintheopen/2007/11/18/an-experiment-in-open-notebook-science-sortase-mediated-protein-dna-ligation/
I am putting up data that we have on a method for the attachment of oligonucleotides to DNA. This data is from experiments carried out by Lilyan Chan who is a student in my lab who is finishing up and therefore has not transferred to the open notebook.

Sortase was prepared by Lilyan Chan in our laboratory. GG-DNA GG-PET2 was obtained from atdbio (www.atdbio.com). EGFP-LPETGG-His6 was prepared in our lab. All reactions were carried out in Sortase buffer.

Experiment 1: Optimising target protein concentration

GG-DNA was ligated to EGFP-LPETGG-His6 protein using sortase A. Different concentrations of EGFP were used to find out optimal concentration to use in the future. Lane 1: Protein marker, lane 2: 0 μM EGFP, lane 3: 1 μM EGFP, lane 4: 5 μM EGFP, lane 5: 10 μM EGFP, lane 6: 20 μM EGFP, lane 7: 50 μM EGFP, lane 8: 75 μM EGFP, lane 9: 100 μM EGFP, lane 10: 150 μM EGFP, lane 11: 200 μM EGFP.


Experiment 2: Changing pH and DNA concentration

GG-DNA was ligated to EGFP-LPETGG-His6 (20 μM, from lane 2 to lane 13) and EGFP-His6 (20 μM, for lane 14 and 15) proteins using sortase A. Different pH of buffer (50 mM Tris, 100 mM NaCl, 5 mM CaCl2) and concentration of DNA was used to find out optimum condition to use in the future. Lane 1: Protein marker, lane 2: 0 μM GG-DNA at pH 7.5, lane 3: 0 μM GG-DNA at pH 9.0, lane 4: 1 μM GG-DNA at pH 7.5, lane 5: 1 μM GG-DNA at pH 9.0, lane 6: 10 μM GG-DNA at pH 7.5, lane 7: 10 μM GG-DNA at pH 9.0, lane 8: 20 μM GG-DNA at pH 7.5, lane 9: 20 μM GG-DNA at pH 9.0, lane 10: 40 μM GG-DNA at pH 7.5, lane 11: 40 μM GG-DNA at pH 9.0, lane 12: 60 μM GG-DNA at pH 7.5, lane 13: 60 μM GG-DNA at pH 9.0, lane 14: EGFP with 20 μM GG-DNA at pH 7.5, lane 15: 20 μM GG-DNA at pH 9.0.

Experiment 3: Changing [Sortase]

Different concentration of EGFP was used to ligate GG-DAN to EGFP to find out optimal concentration to use in the future. Lane 1: Protein marker, lane 2: 0 nM, lane 3: 10 nM, lane 4: 20 nM, lane 5: 40 nM, lane 6: 60 nM, lane 7: 80 nM, lane 8: 100 nM, lane 9: 150 nM, lane 10: 200 nM, lane 11: 250 nM, lane 12: 500 nM, lane 13: 1000 nM.