Difference between revisions of "Part:BBa K3739012"
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===Biology=== | ===Biology=== | ||
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The RhlA comes from Pseudomonas aeruginosa, and is capable of competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the β-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates, therefore supplies the acyl moieties for rhamnolipid biosynthesis. RhlA has a greater affinity for 10-carbon substrates, and is the only enzyme required to generate the lipid component of rhamnolipid.¹ | The RhlA comes from Pseudomonas aeruginosa, and is capable of competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the β-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates, therefore supplies the acyl moieties for rhamnolipid biosynthesis. RhlA has a greater affinity for 10-carbon substrates, and is the only enzyme required to generate the lipid component of rhamnolipid.¹ | ||
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===Usage=== | ===Usage=== | ||
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In order to obtain purified RhlA, we add a his-tag (6∗His) at the N-terminal of RhlA. We used <partinfo>BBa_K081005</partinfo> to construct the expression system and obtained the composite <partinfo>BBa_K3739046</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. | In order to obtain purified RhlA, we add a his-tag (6∗His) at the N-terminal of RhlA. We used <partinfo>BBa_K081005</partinfo> to construct the expression system and obtained the composite <partinfo>BBa_K3739046</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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===Characterization=== | ===Characterization=== | ||
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====1. Agarose Gel Electrophoresis==== | ====1. Agarose Gel Electrophoresis==== | ||
| − | After transferring the plasmid into ''Vibrio natriegens'', colony PCR was used to certify the plasmid was correct. The expected result was obtained(1305bp). | + | After transferring the plasmid into ''Vibrio natriegens'', colony PCR was used to certify the plasmid was correct. The expected result was obtained(1305bp).<br/> |
| − | + | [[File:T--XMU-China--12F1-1.png|300px]]<br/> | |
| − | Fig.1 The result of colony PCR. Plasmid pET-28a(+). | + | '''Fig.1''' The result of colony PCR. Plasmid pET-28a(+). |
===Reference=== | ===Reference=== | ||
Latest revision as of 22:27, 21 October 2021
his-rhlA
Required for rhamnolipid surfactant production.
Biology
The RhlA comes from Pseudomonas aeruginosa, and is capable of competing with the enzymes of the type II fatty acid synthase (FASII) cycle for the β-hydroxyacyl-acyl carrier protein (ACP) pathway intermediates, therefore supplies the acyl moieties for rhamnolipid biosynthesis. RhlA has a greater affinity for 10-carbon substrates, and is the only enzyme required to generate the lipid component of rhamnolipid.¹
Usage
In order to obtain purified RhlA, we add a his-tag (6∗His) at the N-terminal of RhlA. We used BBa_K081005 to construct the expression system and obtained the composite BBa_K3739046, 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 transferring the plasmid into Vibrio natriegens, colony PCR was used to certify the plasmid was correct. The expected result was obtained(1305bp).
Fig.1 The result of colony PCR. Plasmid pET-28a(+).
Reference
1. Zhu, K.; Rock, C. O., RhlA converts beta-hydroxyacyl-acyl carrier protein intermediates in fatty acid synthesis to the beta-hydroxydecanoyl-beta-hydroxydecanoate component of rhamnolipids in Pseudomonas aeruginosa. J Bacteriol 2008, 190 (9), 3147-54.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 87
Illegal XhoI site found at 823 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 312
Illegal BsaI.rc site found at 496