Part:BBa_K1076005
FadL, Long-chain fatty acid outer membrane transporte
Gene of fatty acid transport protein, outer membrane; long-chain fatty acid receptor. We wanted this ORF gene in order to enhance the long chain fatty acids intake by Shewanella putrefaciens to increase B-oxidation and therefore ATP and H+ production.Must be completed with promoter, RBS and terminator.
RDFZ-CHINA-2023
Description
The pathology of acne that fatty acids produced by C.acne result in inflammation and the formation of scar, we decided to design a metabolic pathway to break down these fatty acid using the fadl gene. This gene encodes for FadL which locates in the outer membrane and possesses the ability to facilitate uptake of fatty acids into bacterial body. When this protein binds with fatty acids it undergoes a conformational change which expose the transport channel and facilitate LCFA transport into the periplasmic space, therefore achieving our objective.
Usage and Biology
In this study, the fadL gene was cloned into the pET23b vector to create the recombinant plasmid pET23b-fadL, which was then transformed into E. coli Rosetta.
Figure 1 Design of the fadL.
Characterization
The transformed E. coli Rosetta strains were inoculated into LB medium containing antibiotics and cultured overnight. The optical density at 600 nm (OD600) was adjusted to 1, and 2 mL of bacterial culture was collected by centrifugation at 10,000 rpm for 1 min. Rosetta containing empty plasmid was used as control. The bacterial pellet was resuspended in 2 mL of PBS buffer containing 100 μM palmitic acid and incubated for 1 hour to allow the bacteria to take up the palmitic acid. The bacteria were then collected by centrifugation at 10,000 rpm for 1 min, and the supernatant (extracellular sample) was collected. The bacterial pellet was resuspended in PBS and lysed by sonication at 75W for 5 min to release the intracellular contents (intracellular sample). Lipids were extracted from both extracellular and intracellular samples using the following method: an equal volume of chloroform/methanol (2:1, v/v) was added to the samples, mixed thoroughly, and allowed to stand for 10 minutes to transfer the lipids to the chloroform phase. The samples were then centrifuged at 15,000 rpm for 10 min to separate the chloroform and aqueous phases. The chloroform phase, containing the lipids, was collected and transferred to a new centrifuge tube. The chloroform was evaporated at 50°C in a fume hood and further dried in a vacuum rotary evaporator for 30 minutes. The dried lipids were dissolved in 200 µL of fatty acid assay buffer by vortexing for 5 minutes. As shown in the figure, this is a comparison chart of the intake levels of long-chain fatty acids. The three groups of data represent endocellular, extracellular, and total amounts. By comparing with the control group, it can be concluded that FadL enhances the intake of long-chain fatty acids overall, especially in the extracellular compartment.
Fig 2 FadL promotes the uptake of long chain fatty acids.
Potential application directions
This experiment demonstrates that the overexpression of fadl facilitates the ability of fatty acid uptake for e.coli, revealing a possibility to regulate extracellular fatty acid. Providing a solution to the current pharmaceutical vacancy for a drug that assists the metabolism of fatty acid.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 716
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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