Difference between revisions of "Part:BBa K3196020"
GlacierHOLE (Talk | contribs) |
|||
| (4 intermediate revisions by 2 users not shown) | |||
| Line 6: | Line 6: | ||
<h1>'''Characterization'''</h1> | <h1>'''Characterization'''</h1> | ||
| − | This is a four section for degrade and transfer | + | This is a four section for degrade and transfer pectin part. |
| − | [[File: | + | [[File:T--HUST--China--2019-SUC2pelA.jpg |400px|thumb|center|Figure1. T--HUST--China--2019-SUC2pelA]] |
<h1>'''DNA Gel Electrophoretic'''</h1> | <h1>'''DNA Gel Electrophoretic'''</h1> | ||
| − | After we link SUC2 and pelA successfully, we run the PCR with an intention to confirm the expression of pelA. As the figure shows, we get the genetic stripe about | + | After we link SUC2 and pelA successfully, we run the PCR with an intention to confirm the expression of pelA. As the figure shows, we get the genetic stripe about 1092 bp which means the PCR is successful. |
| − | + | Electro-transformation to Pichia pastoris was all succeeded. | |
| − | + | [[File:T--HUST--China--2019-pelA_Gel.png|400px|thumb|center| Figure1 Yeast Genome extraction and pcr]] | |
| − | + | ||
| − | + | ||
| − | [[File: | + | |
<h1>'''Enzyme Activity'''</h1> | <h1>'''Enzyme Activity'''</h1> | ||
| − | + | The enzyme activity of the pelA group is obtained by titration and daily measurement of enzyme activity. | |
| − | [[File: | + | [[File:T--HUST-China--2019-pelA data.jpg |400px|thumb|center| Figure2 Determination of pelA enzyme activity]] |
| + | |||
| + | |||
| + | Pectin is a hydrophilic polysaccharide substance present in the interstitial of plant cells. It is stable in acidic medium and insoluble in ethanol. It is a natural polymer compound. We understand that pectinase measures enzyme activity has a nationally standardized experimental procedure, so we have adopted the standard titration scheme given by the state. <sup> [3] </sup> | ||
| + | |||
| + | |||
| + | |||
<!-- --> | <!-- --> | ||
Latest revision as of 11:37, 20 October 2019
AOX1-Kozak-SUC2-pelA-His tag-AOX1 Terminator
The SUC2 gene of S. cerevisiae, encoding invertase was designed to codon preference of P. pastoris excluding the sequence of the N-terminal pre-pro-sequence signal peptide and His-tag in the C terminus. pelA can catalyze pectin.
Characterization
This is a four section for degrade and transfer pectin part.
DNA Gel Electrophoretic
After we link SUC2 and pelA successfully, we run the PCR with an intention to confirm the expression of pelA. As the figure shows, we get the genetic stripe about 1092 bp which means the PCR is successful. Electro-transformation to Pichia pastoris was all succeeded.
Enzyme Activity
The enzyme activity of the pelA group is obtained by titration and daily measurement of enzyme activity.
Pectin is a hydrophilic polysaccharide substance present in the interstitial of plant cells. It is stable in acidic medium and insoluble in ethanol. It is a natural polymer compound. We understand that pectinase measures enzyme activity has a nationally standardized experimental procedure, so we have adopted the standard titration scheme given by the state. [3]
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1761
Illegal BamHI site found at 937 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]