Difference between revisions of "Part:BBa K5175014"
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<partinfo>BBa_K5175014 parameters</partinfo> | <partinfo>BBa_K5175014 parameters</partinfo> | ||
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| + | <h1>'''Usage and Biology'''</h1> | ||
| + | PHA are a class of biopolyesters with plastic-like properties produced by microorganisms under nutrient-limited conditions [21]. P.putida synthesizes PHA via the fatty acid β-oxidation pathway and fatty acid de novo synthesis pathway, which serves as an intracellular storage material for energy and carbon sources. Whereas during PHA synthesis, it competes with rhamnolipid synthesis for the same substrate, β-hydroxyl-ACP. | ||
| + | In order to increase the proportion of the carbon source utilised by P.putida that flows to the rhamnolipid synthesis pathway, we overexpressed the gene encoding the poly(3-hydroxyalkanoate) depolymerase gene phaZ. thus, inhibiting PHA anabolic bypass increases rhamnolipid production. | ||
| + | |||
| + | <h1>'''Molecular cloning'''</h1> | ||
| + | In order to construct the desired plasmids, we first perform double enzyme digestion to obtain multiple fragments and vectors with the same sticky ends, and then ligate the vectors and fragments containing the same sticky ends together by performing ligation. Finally, the complete plasmid was introduced into <i>P. putida</i> KT2440 cells by using electroporation, and their | ||
| + | successful integration was verified through colony PCR analysis. | ||
Revision as of 22:33, 1 October 2024
RBS for T7 promoter
This is the RBS for T7 promoter.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
PHA are a class of biopolyesters with plastic-like properties produced by microorganisms under nutrient-limited conditions [21]. P.putida synthesizes PHA via the fatty acid β-oxidation pathway and fatty acid de novo synthesis pathway, which serves as an intracellular storage material for energy and carbon sources. Whereas during PHA synthesis, it competes with rhamnolipid synthesis for the same substrate, β-hydroxyl-ACP. In order to increase the proportion of the carbon source utilised by P.putida that flows to the rhamnolipid synthesis pathway, we overexpressed the gene encoding the poly(3-hydroxyalkanoate) depolymerase gene phaZ. thus, inhibiting PHA anabolic bypass increases rhamnolipid production.
Molecular cloning
In order to construct the desired plasmids, we first perform double enzyme digestion to obtain multiple fragments and vectors with the same sticky ends, and then ligate the vectors and fragments containing the same sticky ends together by performing ligation. Finally, the complete plasmid was introduced into P. putida KT2440 cells by using electroporation, and their successful integration was verified through colony PCR analysis.