Difference between revisions of "Part:BBa K3196011"
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<h1>'''DNA Gel Electrophoretic'''</h1> | <h1>'''DNA Gel Electrophoretic'''</h1> | ||
− | After we link FLO10 and SLAC successfully, we run the PCR with an intention to confirm the expression of SLAC. As the figure shows, we get the genetic stripe about | + | After we link FLO10 and SLAC successfully, we run the PCR with an intention to confirm the expression of SLAC. As the figure shows, we get the genetic stripe about 2009 bp which means the PCR is successful. |
− | [[File: | + | [[File:T--HUST-China--2019-DNA Gel Electrophoretic.png|400px|thumb|center|Figure1:This is the result of the SacⅠ single endonuclease digestion plasmid of the TOP10 strain, we will extract the gel and obtain the aimed DNA. ]] |
<h1>'''SDS-PAGE'''</h1> | <h1>'''SDS-PAGE'''</h1> | ||
− | We run the SDS-PAGE to check whether FLO10 help the enzyme transfer to the extracellular. As the figure shows, we get the protein type about | + | We run the SDS-PAGE to check whether FLO10 help the enzyme transfer to the extracellular. As the figure shows, we get the protein type about 35 KDa which means the SDS-PAGE is successful. |
− | [[File: | + | [[File:T--HUST-China--2019-SLAC-SDS-PAGE.jpg |400px|thumb|center|Figure3:the SDS-page result shows that Pichia pastoris have successfully secrete SLAC protein into superfluous liquid.]] |
+ | |||
<h1>'''Enzyme Activity'''</h1> | <h1>'''Enzyme Activity'''</h1> | ||
We use ABTS to detect the enzyme activity. As the figure shows, the solution turns green, which confirm the enzyme activity. | We use ABTS to detect the enzyme activity. As the figure shows, the solution turns green, which confirm the enzyme activity. | ||
− | [[File: | + | [[File:T--HUST-China--2019-SLAC enzyme activity.png|400px|thumb|center|Figure4:these four pictures shows the enzyme activity changes with the time. The four kinds of Pichia pastoris have the different curve, and the most activity strain is FLO10-apro, PHO5apro shows a more delayed increase on enzyme activity.]] |
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Revision as of 05:56, 20 October 2019
AOX1-Kozak-FLO10-SLAC-His tag-AOX1 Terminator
FLO10 is a Lectin-like protein with similarity to Flo1 p, thought to be expressed and involved in flocculation. SLAC can catalyze lignin.
Characterization
This is a four section for degrade and transfer lignin part.
DNA Gel Electrophoretic
After we link FLO10 and SLAC successfully, we run the PCR with an intention to confirm the expression of SLAC. As the figure shows, we get the genetic stripe about 2009 bp which means the PCR is successful.
SDS-PAGE
We run the SDS-PAGE to check whether FLO10 help the enzyme transfer to the extracellular. As the figure shows, we get the protein type about 35 KDa which means the SDS-PAGE is successful.
Enzyme Activity
We use ABTS to detect the enzyme activity. As the figure shows, the solution turns green, which confirm the enzyme activity.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 937
Illegal BamHI site found at 948 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1387