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6?g nicotinic acid h?1?g?1DCW and 21?g INA h?1?g?1DCW were achieved respectively. These work established a solid step towards commercialization. Genetic studies of Nocardia were greatly hampered for a long period because of the lack of genetic tools including efficient DNA transfer methods. In 1994, Yao and his colleagues developed the first cloning vector pCY104 for Nocardia (Yao et?al., 1994). It employed a Nocardia-derived cryptic plasmid pCY101 as replication origin and three resistance genes from a plasmid used in Escherichia coli for genetic selection. In consideration of the large size (8.9?kb), pCY104 was not extensively used. In recent years, a new series of Nocardia-E.?coli vectors (named pNV) employing a mycobacterial plasmid pAL5000 as replication origin were constructed and improved (Ishikawa et?al., 2006; Chiba et?al., 2007). The www.selleckchem.com/products/gw3965.html current versions pNV118 and pNV119 have Perifosine ic50 many desirable features for practical manipulations (Hoshino et?al., 2010): small size (4.4?kb), multiple cloning site, blue-white selection in E.?coli, kanamycin and neomycin resistance, high copy number and broad host-range. In addition, the Rhodococcus, Gordonia-E.?coli shuttle vector pNC9503 (Kalscheuer et?al., 1999) also showed the ability to replicate in Nocardia (Luo et?al., 2013). The pNC903-derived replication origin and the thiostrepton resistance on pNC9503 make it an alternative choice to the pNV series vectors. Because of the thick cell wall structure of Gram-positive bacteria, electroporation has become a powerful tool to introduce foreign DNA into these cells. This is also the case for Nocardia. Yao and colleagues reported an electroporation efficiency of 8?��?104 colony-forming units (CFUs)?��g�C1 plasmid DNA by using pCY104 in N.?asteroides YP21. However, plasmids must be prepared eltoprazine from N.?asteroids; plasmids derived from E.?coli gave a 102�C103-fold lower efficiency (Yao et?al., 1994). Maharjan and colleagues (2012) studied the effects of growth temperature and field strength on electroporation efficiency. Unfortunately, the obtained efficiency (