Berkeley LBL/Methods
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Experimental
Experimental
Subcloning
The genes of interest from Rhodobacter sphaeroides, Synechocystis sp, and Heliobacillus mobilis were amplified using PCR (the details of which can be found in Protocols). The PCR fragments were then digested and ligated with the T7 expression vector pET3a in various ways, resulting in the constructs shown below. They were transformed into E.Coli (either DH10B or NovaBlue) and glycerol stocks for these cells were saved.
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
Expression
Once the constructs are inside DH10B or NovaBlue, they were minipreped and transformed in BL21 (DE3). These cells were then induced to overexpress the proteins of interest. After protein expression, the cell cultures were centrifuged at 10,000rpm for 20 minutes. The resulting cell pellets were dissolved in TRIS buffer (pH 7.8) and sonicated. The sonicated cell extracts were then centrifuged again, with the supernatant containing the soluble proteins. The soluble protein extract was used for enzyme activity assays. Along with the soluble protein extract, the total sonicated cell extract and the insoluble cell extract were analyzed on a SDS-PAGE gel.
Enzyme Activity Assays
The E.Coli cells with the constructs pET3a-R-bchHID and pET3a-S-bchHID have all the peptides necessary for the enzyme Mg-chelatase. The activity of these enzymes can be measured by the concentration of the reaction product, Mg-protoporphyrin IX. The concentrations were measured by the UV-vis spectroscopy and fluorescence emission spectroscopy, utilizing the Beer-Lambert law. By assuming that the concentration of the product is proportional to the activity of the enzymes, we can determine which enzyme (either from Rhodobacter sphaeroides or Synechocystis sp) is more efficient.
Protocols
PCR (Using Phusion Polymerase)
PCR (Using TaKaRa Ex Taq Polymerase)
Digestion for PCR Product or Miniprepped DNA
KCM Competent Cell Transformation
Electroporation Transformation
