Difference between revisions of "Part:BBa K861001"

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Ampicilin (100 mg/mL) was prepared and added to LB medium, E. coli was inoculated into LB medium, and the plates were incubated overnight in a shaker.

Plasmids were extracted for DH5α/pSB1A3-mRFP and DH5α/pSB1A3-insert previously and digested. Colonies were screened by PCR after this

DNA was purified from agarose gels and the heavier bands in the RFP column and the lighter bands in the Insert column were extracted to obtain the RFP vector and Insert gene.

After synthesizing plasid PCR, competent cells were prepared from E. coli DH5α and E. coli BL21 and screened for colonies by PCR. Half of the DH5α colonies were added to the PCR mixture as template and PCR was performed.

 Agarose gel electrophoresis was performed on PCR products.

2mL of bacterial broth was extracted from LB liquid medium. The plasmid was extracted from the bacterial broth to obtain the validated correct DH5α/pSB1A3-GFP. and cultured in a shaker.

Extract 2mL of bacterial solution from LB liquid medium. Extract the plasmid from the bacterial solution to obtain the validated correct DH5α/pSB1A3-GFP. Measure the concentration and OD260/OD280 of the plasmid in the extraction solution. collect the prepared E. coli BL21 cells, and then transfer the pSB1A3-GFP into the bacterial cells. The BL21 transformants were placed in AMP LB liquid medium and shaken overnight. Fluorescence microscopy verification experiments were performed to verify whether the pSB1A3-GFP gene was successfully inserted and whether the plasmid was successfully transferred into BL21 cells.

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Test:

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Our experiments found that the extracellular concentration of palmitic acid decreased to nearly half when the strain contained the FadL gene compared to the control; while the intra-package concentration increased. This indicates that the purpose of our strain design, i.e., to accelerate the rate of palmitic acid transport by probiotics increased.

FadL with a strong RBS and a double terminator

Long-chain fatty acids are transported across the cell membrane via a transportation/acyl-activation mechanism involving an outer membrane protein, FadL. The FadL gene encodes a 43,000-dalton membrane protein (FLP) which has been implicated as being essential for LCFA transportation. When expressed, FadL can transport the long-chain fatty acids into the bacterium.

BBa_K861001 contains coding sequence of FadL with a strong RBS and double terminators

Sequence and Features