Cloning and expression of metallothionein mutant alpha-KKS-alpha in Anabaena sp. PCC 7120.
The mouse metallothionein (mMT) mutant alpha-KKS-alpha has a higher capacity for binding heavy metals than wild type mMT. The mMT mutant alpha-KKS-alpha gene was placed under the control of the strong promoter PpbsA to generate the intermediate vector pRL-alpha-KKS-alpha. pRLalpha-KKS-alpha was then linked with the plasmid pDC-08 to construct shuttle expression vector pDC-alphaKKS-alpha. This expression vector was transformed into Anabaena sp. PCC 7120 using triparental conjugative transfer. After antibiotic selection (ampicillin and kanamycin), transgenic Anabaena was identified by PCR and Western blotting. The expression level of the mMT mutation alpha-KKS-alpha reached 7.4 mg/g dry cells weight, as detected by ELISA, and heavy metal resistance of the transgenic Anabaena was significantly improved.
Shao
Qiang
Q
College of Life Sciences, Peking University, Beijing, PR China.
Shi
Ding-Ji
DJ
Hao
Fu-Ying
FY
Ma
Li-Na
LN
Chen
Zhen-Jia
ZJ
Yu
Mei-Min
MM
Ru
Bing-Gen
BG
D017033:Anabaena / Q000235:genetics* / Q000502:physiology; D015153:Blotting, Western; D003001:Cloning, Molecular; D004351:Drug Resistance; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D008668:Metallothionein / Q000096:biosynthesis* / Q000235:genetics*; D019216:Metals, Heavy / Q000494:pharmacology*; D010957:Plasmids; D016133:Polymerase Chain Reaction
D019216:Metals, Heavy; D008668:Metallothionein