Part:BBa_K2275009
PlacI-B0034-glnA
This composite part is composed of R0010, B0034 and K2275003 which encodes glutamate dehydrogenase (GudB) derived from Pseudomonas putida, having the ability to catalyze glutamate to glutamine. First, to ensure whether the gene glnA can successfully express in E. coli or not, we used SDS-PAGE method to analyze the protein expression of GlnA/MG1655. The SDS-PAGE result is shown below. "WC" indicates whole cell, "S" for supernatant, "P" for pellet, and "C" indicates the control of sample without plasmid.
Luckily, the expression of glnA was most in supernatant form. To test if this Biobrick is functionable, we added the glutamate and use Glutamine Colorimetric Assay Kit to detect the glutamine concentration. The assay is based on the hydrolysis of glutamine to glutamate producing a stable signal, which is directly proportional to the amount of Gln in the sample. The process of the assay is shown below.
By adding different concentration of glutamate and detecting the absorbance at OD 450 nm, we can find out the relation of glutamine concentration for corresponding OD at 450 nm. The calibration curve is shown below.
Following, we also do three different experiment to test if the E. coli MG1655 contain the Biobrick: BBa_K2275009 is better to produce more glutamine. The assay result is shown below.
The assay result shows that E. coli harboring the plasmid can convert glutamate into glutamine. The higher concentration of glutamate is, the higher glutamine will produce. It explains that the GlnA can successfully express by E. coli MG1655 and convert glutamate into glutamine.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 675
Illegal BamHI site found at 1309 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 322
Illegal NgoMIV site found at 1088
Illegal AgeI site found at 578 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1481
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