Part:BBa_K5175041
T7 promoter-rhlA-rhlB-T7 terminator
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 827
Illegal PstI site found at 1811
Illegal PstI site found at 1925 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2310
Illegal PstI site found at 827
Illegal PstI site found at 1811
Illegal PstI site found at 1925 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 176
Illegal BamHI site found at 736
Illegal XhoI site found at 912
Illegal XhoI site found at 2159 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 827
Illegal PstI site found at 1811
Illegal PstI site found at 1925 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 827
Illegal PstI site found at 1811
Illegal PstI site found at 1925
Illegal NgoMIV site found at 1052
Illegal NgoMIV site found at 1773
Illegal NgoMIV site found at 1886 - 1000COMPATIBLE WITH RFC[1000]
Description
It is a composite component consisting of the T7 promoter, T7 terminator, target genes rhlA, rhlB. This component is responsible for encoding the Rhamnosyltransferase I in the engineered bacterium P.putida, adding a new exogenous pathway for synthesizing β-hydroxy fatty acids and catalysing the synthesis of mono-rhamnolipids containing one rhamnosyl group from dTDP-L-rhamnose and β-hydroxy fatty acids.
Usage and Biology
rhlA
Rhamnolipid is a biosurface activator first isolated from Pseudomonas aeruginosa by Jarvis and Johnson, Rhamnolipid emulsifies, disperses, and solubilizes hydrophobic organic pollutants and improves their bioavailability, facilitating the removal of hydrophobic organic pollutants. Rhamnolipids also have some metal chelating ability and are used to remove heavy metals from soil, sewage and other liquids. In agricultural applications, rhamnolipids can be used to improve soil, enhance the effects of pesticides and fertilizers, and inhibit agricultural diseases.
Rhamnosyltransferase I is a key enzyme necessary for the synthesis of rhamnolipids, which is a complex enzyme containing 2 subunits, RhlA and RhlB, encoded by the rhlAB gene on the same manoeuvre rhlABRI. Among them, RhlA encoded by the rhlA gene is a phthalyltransferase responsible for the synthesis of β-hydroxy fatty acids.
rhlB
Rhamnolipid is a biosurface activator first isolated from Pseudomonas aeruginosa by Jarvis and Johnson, Rhamnolipid emulsifies, disperses, and solubilizes hydrophobic organic pollutants and improves their bioavailability, facilitating the removal of hydrophobic organic pollutants. Rhamnolipids also have some metal chelating ability and are used to remove heavy metals from soil, sewage and other liquids. In agricultural applications, rhamnolipids can be used to improve soil, enhance the effects of pesticides and fertilizers, and inhibit agricultural diseases.
Rhamnosyltransferase I is a key enzyme necessary for the synthesis of rhamnolipids, which is a complex enzyme containing 2 subunits, RhlA and RhlB, encoded by the rhlAB gene on the same manoeuvre rhlABRI. Among them, RhlB encoded by the rhlB gene is responsible for catalyzing the synthesis of mono-rhamnolipids containing one rhamnose group from dTDP-L-rhamnose and β-hydroxy fatty acids.
Molecular cloning
In order to construct the desired plasmids, we employed the E.coli DH5α amplification method. Firstly, we performed PCR amplification using specific primers for each plasmid, which results in the generation of linearized fragments harboring the target sequences in a high copy number. These fragments were then connected into complete plasmids using enzyme-cutting and enzyme-linking procedures. After transfer to Escherichia coli, colony PCR was used to confirm successful construction of the plasmid. Subsequently, the plasmids were further amplified to obtain sufficient quantities for further experiments. Finally, The plasmids were successfully introduced into P.putida KT2440 through electroporation. Given that our wild-type P.putida KT2440 exhibits resistance to chloramphenicol, the plasmids incorporated a kanamycin resistance marker. Consequently, we employed dual antibiotic selection plates to effectively screen for successfully transformed engineered strains.
The bands of rhlA-rhlB（2000+ bp）from PCR are identical to the theoretical lengths of 2452bp estimated by the designed primer locations (promoter to terminator), which could demonstrate that these plasmids had successfully been obtained.
The sulfuric acid-anthrone method is capable of quantifying the total sugar content in the sample solution. Rhamnolipids, functioning as surfactants, consist of both reducing sugar and long-chain fatty acid components. Organic solvent extraction can remove soluble sugars from the fermentation broth. In our experiments, P.putida KT2440 (rhlA-rhlB) exhibited significantly higher rhamnolipid production compared to the wild-type strain. However, since the wild-type also produced a measurable amount, we planned to design further experiments, including mass spectrometry, to confirm the production of rhamnolipids. Unfortunately, due to time constraints, we were unable to successfully detect rhamnolipids.
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