Difference between revisions of "Part:BBa K2275008"

Latest revision as of 07:36, 28 October 2017

PlacI-B0034-gudB

This composite part is composed of R0010, B0034 and K2275002 which encodes glutamate dehydrogenase (GudB) derived from Bacillus subtilis, having the ability to transform ammonium to glutamate. First, to ensure whether the gene gudB can successfully express in E. coli or not, we used SDS-PAGE method to analyze the protein expression of GudB/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.

SDS-PAGE result shows that most protein is in form of pellet. Compared to the control, the expression level was much higher. However, no supernatant protein doesn't mean that it has no function. Hence, we added the ammonium and use Glutamate Colorimetric Assay Kit to detect the glutamate concentration. The Glutamate Enzyme Mix recognizes glutamate as a specific substrate and lead to proportional color development. The amount of glutamate can therefore be easily quantified by spectrophotometry at 450 nm. First, calibration curve is desired. By adding different concentration of glutamate and detecting the absorbance at 450 nm, we plot the relation between OD and glutamate concentration. The data of calibration curve is shown below.

The next step, we do three different experiment to test if the E. coli MG1655 harboring the Biobrick: BBa_K2275009 can convert the ammonium into glutamate. The testing result is shown below.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 682
Illegal AgeI site found at 245
1000
COMPATIBLE WITH RFC[1000]

@@ Line 1: / Line 1: @@
 __NOTOC__
 <partinfo>BBa_K2275008 short</partinfo>
-This composite part is composed of R0010, B0034 and K2275002 which encodes glutamate dehydrogenase (GudB) derived from Bacillus subtilis. First, to ensure whether the gene gudB can successfully express in E. coli or not, we used the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to analyze the protein expression of GudB. The SDS-PAGE result is shown below.
+This composite part is composed of R0010, B0034 and K2275002 which encodes glutamate dehydrogenase (GudB) derived from Bacillus subtilis, having the ability to transform ammonium to glutamate. First, to ensure whether the gene gudB can successfully express in E. coli or not, we used SDS-PAGE method to analyze the protein expression of GudB/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.
-[[File:SDS-PAGE_of_GudB.png|350px||centre]]
+[[File:SDS-PAGE result of GudB.png|220px||centre]]
+SDS-PAGE result shows that most protein is in form of pellet. Compared to the control, the expression level was much higher. However, no supernatant protein doesn't mean that it has no function. Hence, we added the ammonium and use Glutamate Colorimetric Assay Kit to detect the glutamate concentration. The Glutamate Enzyme Mix recognizes glutamate as a specific substrate and lead to proportional color development. The amount of glutamate can therefore be easily quantified by spectrophotometry at 450 nm. First, calibration curve is desired. By adding different concentration of glutamate and detecting the absorbance at 450 nm, we plot the relation between OD and glutamate concentration. The data of calibration curve is shown below.
+[[File:Glutamate_calibration_curve.png|500px||centre]]
+The next step, we do three different experiment to test if the E. coli MG1655 harboring the Biobrick: BBa_K2275009 can convert the ammonium into glutamate. The testing result is shown below.
+[[File:Glutamate_functional_test.png|450px||centre]]
 <!-- Add more about the biology of this part here