Berkeley LBL/Methods

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Experimental

Constructs

The T7 expression vector pET3a was used for the subcloning of the genes for magnesium-chelatase. The following constructs were built by inserting various fragments genes from the three organisms into pET3a. (Note: The pET3a vector contains a ribosome binding site downstream of the T7 promoter region; this rbs would not be indicated below)

Magnesium-Chelatase
Rhodobacter sphaeroides
pET3a-bchH
pET3a-bchI
pET3a-bchD
pET3a-bchHID
Synechocystis sp.
pET3a-chlH
pET3a-chlI
pET3a-chlD
pET3a-chlHID

Heliobacillus mobilis

Protocols

PCR (Using Phusion Polymerase)

PCR (Using TaKaRa Ex Taq Polymerase)

PCR Clean Up/Purification

DNA Gel Electrophoresis

Miniprep

Digestion for PCR Product or Miniprepped DNA

Analytic Digestion

Gel Extraction

Ligation

KCM Competent Cell Production

KCM Competent Cell Transformation

Electroporation Transformation

Overexpression

Sonication

SDS-PAGE