Continuing previous experiment, today I’m going to transform gene encoding phytase from aspergillus oryzae, Aspergillus niger, GFP+S1 (we mutated this gene through PCR and cloned into vector pGEMT easy, amazingly It glows up) into E.coli DH5alpha.
this morning we have subcultured E.coli 1% of total volume (1% of 25 ml=250uL) into fresh LB 25 ml, then incubated at 37 Celcius degree for about 3 hours. after 3 hour we prepared for making competent cells, heat shock, recovery cell, and plating onto Luria Agar containing antibiotic resistance.
if your protein is cytosolic and non-glycosilated, you may elect to express the protein intracellularly using one of the PPICZ vectors. PPICZ A,B,C (www.invitrogen.com) well-known are expression vector in yeast especially P. pastoris.
however if you wish your protein expressed outer cell membrane,glycosilated, or directed into an intracellular organelle, you may wish to try secreting your protein using on of the PPICZ alpha vector. invitrogene had recommended us to try both the native secretion signal and alpha-factor signal sequence in order to secrete our protein.
Finishing transformation is to plate e.coli recovered-cells onto appropriate media.
1. pOKBAD4-Sp/Sm (LA+Km 50+Sp25) 6. PPICZB-GFP+S1 EcoRI (LA+Zc 25)
2. pOKBAD9-Sp/Sm (LA+Km 50+Sp25) 7. PPICZ-alpha -AF new (LA+Zc 25)
3. pOKBAD13-Sp/Sm (LA+Km 50+Sp25) 8. PPICZ-alpha -AO1 (LA+Zc 25)
4. pOK-E.coli hemB-Gm (LA+Km 50+Gm 10) 9. PPICZ-alpha -AO5 (LA+Zc 25)
5. pGLO-GFP+S1 EcoRI (LA+Ap100+Ara 0.2%) 10. PPICZ-alpha -AO6 (LA+Zc 25)
Maps of PPICZ can be viewed at www.genomex.com/vector_maps/ppicz_map.pdf
