Difference between revisions of "Part:BBa K3739013"

Latest revision as of 22:31, 21 October 2021

his-rhlB

transferase activity, transferring hexosyl groups.

Biology

RhlB

The rhamnosyltransferase (RhlB) comes from Pseudomonas aeruginosa, is capable of catalyzing the reaction between HAA and dTDP-l-rhamnose to form mono-rhamnolipid.1 The hydropathy plot of the RhlB suggested that the protein is 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 correctness of the plasmid. Target bands (1698 bp) can be observed at the position around 1700 bp (Fig. 1).

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

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 1156
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 49
Illegal NgoMIV site found at 883
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 399

@@ Line 9: / Line 9: @@
 ====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&sup1;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. &sup2;
+The rhamnosyltransferase (RhlB) comes from ''Pseudomonas aeruginosa'', is capable of catalyzing the reaction between HAA and dTDP-l-rhamnose to form mono-rhamnolipid.1 The hydropathy plot of the RhlB suggested that the protein is 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&lowast;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&lowast;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.
@@ Line 19: / Line 18: @@
 . Agarose Gel Electrophoresis
-After receiving the plasmid from Sangon Biotech&reg;, regular PCR was used to certify the plasmid was correct. The expected result was obtained(1698bp).
+After receiving the plasmid from Sangon Biotech®, regular PCR was used to certify the correctness of the plasmid. Target bands (1698 bp) can be observed at the position around 1700 bp (Fig. 1).<br/>
-Fig.1 The result of regular PCR. Plasmid pET-28a(+).
+[[File:T--XMU-China--13-F1.png|300px]]<br/>
+'''Fig.1''' The result of regular PCR. Plasmid pET-28a(+).
 ====Reference====
-.	Chong, H.; Li, Q., Microbial production of rhamnolipids: opportunities, challenges and strategies. Microb Cell Fact 2017, 16 (1), 137.
+.	Chong, H.; Li, Q., Microbial production of rhamnolipids: opportunities, challenges and strategies. ''Microb Cell Fact'' '''2017''', 16 (1), 137.
-.	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.
+.	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.