Difference between revisions of "Part:BBa K3739013"
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====Usage==== | ====Usage==== | ||
| − | In order to obtain purified RhlB, we added a his-tag (6∗His) at the N-terminal of RhlB. We used BBa_K081005 to construct the expression system and obtained the composite BBa_K3739047, which is assembled on the expression vector pET-28a(+) by standard assembly. The constructed plasmid was transformed into ''Vibrio natriegens'', then the positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing. | + | In order to obtain purified RhlB, we added a his-tag (6∗His) at the N-terminal of RhlB. We used <partinfo>BBa_K081005</partinfo> to construct the expression system and obtained the composite <partinfo>BBa_K3739047</partinfo>, which is assembled on the expression vector pET-28a(+) by standard assembly. The constructed plasmid was transformed into ''Vibrio natriegens'', then the positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing. |
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1. Chong, H.; Li, Q., Microbial production of rhamnolipids: opportunities, challenges and strategies. Microb Cell Fact 2017, 16 (1), 137. | 1. Chong, H.; Li, Q., Microbial production of rhamnolipids: opportunities, challenges and strategies. Microb Cell Fact 2017, 16 (1), 137. | ||
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2. Ochsner, U. A.; Fiechter, A.; Reiser, J., Isolation, characterization, and expression in Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase involved in rhamnolipid biosurfactant synthesis. J Biol Chem 1994, 269 (31), 19787-19795. | 2. Ochsner, U. A.; Fiechter, A.; Reiser, J., Isolation, characterization, and expression in Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase involved in rhamnolipid biosurfactant synthesis. J Biol Chem 1994, 269 (31), 19787-19795. | ||
Revision as of 16:27, 20 October 2021
his-rhlB
transferase activity, transferring hexosyl groups.
Biology
RhlB
The RhlB comes from Pseudomonas aeruginosa, and is a rhamnosyltransferase that is capable of catalyzing the reaction between HAA and dTDP-l-rhamnose to form mono-rhamnolipid.¹ The hydropathy plot of the RhlB suggested the protein to be membrane anchored via its N-terminal, crossing the membrane and exposing its domains on both sides of the membrane. ²
Usage
In order to obtain purified RhlB, we added a his-tag (6∗His) at the N-terminal of RhlB. We used BBa_K081005 to construct the expression system and obtained the composite BBa_K3739047, which is assembled on the expression vector pET-28a(+) by standard assembly. The constructed plasmid was transformed into Vibrio natriegens, then the positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing.
Characterization
1. Agarose Gel Electrophoresis After receiving the plasmid from Sangon Biotech®, regular PCR was used to certify the plasmid was correct. The expected result was obtained(1698bp). Fig.1 The result of regular PCR. Plasmid pET-28a(+).
Reference
1. Chong, H.; Li, Q., Microbial production of rhamnolipids: opportunities, challenges and strategies. Microb Cell Fact 2017, 16 (1), 137.
2. Ochsner, U. A.; Fiechter, A.; Reiser, J., Isolation, characterization, and expression in Escherichia coli of the Pseudomonas aeruginosa rhlAB genes encoding a rhamnosyltransferase involved in rhamnolipid biosurfactant synthesis. J Biol Chem 1994, 269 (31), 19787-19795.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1156
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 49
Illegal NgoMIV site found at 883 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 399