Difference between revisions of "Part:BBa K4202009:Experience"

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Applications of BBa_K4202009

1.Construction of Pet28a-His tag-ACCBP

We obtained the CDS and protein sequence of ACCBP and SacB secretion signal sequence of Bacillus subtilis from NCBI database, and designed the fusion protein SP-ACCBP. The codon of SP-ACCBP was optimized for Bacillus subtilis and synthesized by GenScript, and the plasmid PHY-300PLK-SP-ACCBP(PHY-ACCBP) was constructed.
Considering the large quantity of protein we needed for function verification, we constructed the expression plasmid PET-28a(+)-ACCBP for E.coli in order to obtain the purified protein of high concentration for later experiments. Besides, we also constructed the palsmid Pet28a(+)-Δ16 ACCBP（BBa_K4202010）to explore the influence of N-terminal amino acids to protein folding.

2.Protrein expression verification

To confirm that the optimized sequence could be produced by E. coli, we transformed the plasmid pET-28a (+)-ACCBP into BL21 (DE3) strain and cultured them in kanamycin resistant LB plates at 37 ^o C overnight. The next day, the overnight-cultured solution was transferred into 5 ml kanamycin resistant LB medium at a ratio of 1:20. The solution was cultured at 37°C, 220 rpm until OD₆₀₀ reached 0.6-0.8. Then 0.1 mM IPTG, 10% glycerol and 1mM CaCl₂ was added to induce the protein expression at 8^oC

At the end of induction, the bacterial solution was centrifuged at 12000 rpm at 4°C and the bacteria were collected. After washing the precipitation once with PBS, the precipitation were resuspended in a 1.5ml EP tube with 1mL PBS (appropriate amount of PMSF can be added).The bacteria were disrupted by ultrasonic disruption until the suspension was clear. The products are used for SDS-PAGE.

Result: Acorrding to the result of SDS-PAGE, we can found that the more protein were both in the supernatant and precipitation. And the more protein are in the precipitation, indicating that under this condition majority of the protein are insoluble.

Fig 1 SDS-PAGE and Coomassie brilliantblue staining results of supernatant and sediment of BL21(DE3) strain with PET-28a(+)-ACCBP. The lanes have been indicated .

3.Protein expression and purification

The overnight-cultured bacterial fluid was inoculated into 500 ml Kanamycin resistant LB medium at the ratio of 1:100. The solution was cultured at 37 ^° C , 220rpm until OD₆₀₀ reached 0.6-0.8 and protein was induced at 8 ^oC with 1mM IPTG, 1% glycerol and 1mM CaCl2 for 48h. Then we collect the bacterium and resuspend in a special protein extract buffer (containing 10% glycerol, 500 mM PH 7.5 Tris-HCl buffer, and 0.01mM PMSF ). The next steps are same as 1 above .
Notes: In order to get a better protein expression effect, we also use palsmids Pet28a(+)-ACCBP, Pet28a(+)-Δ16 ACCBP(BBa_K4202010), strains BL21(DE3) and shuffle T7 for this experiment .

Result : The purified ACCBP and Δ16 ACCBP protein were obtained by multiple combination of plasmids and strains. The protein will be used in further experiments.

Fig 2 SDS-PAGE and Coomassie brilliantblue staining results of protein purification. A: The purification result of ACCBP; B: The purification result of Δ16 ACCBP. The results show that both proteins have a considerable abundance in eluent 2 and 3 although there are some non-objective bands shown due to proteolysis.

4.Verification of ACCBP structure

In order to verify the structure of active ACCBP protein, we conducted chemical crosslinking experiment. Add 0.1% and 0.5% glutaraldehyde to 25μl ACCBP protein eluent, mix and let it stand at room temperature. Take samples at regular intervals. After terminating the reaction with loading buffer, run 12% SDS-PAGE to detect the cross-linking structure.

Result: According to the result of SDS-PAGE, we found that that the band of 140kDa was the most significant one of all samples. As the apparent molecular weight of ACCBP monomer is about 25kDa，we predicted that ACCBP probably assembles as pentamers in the solution. And the cross-linking results were better with less non-objective bands when higher glutaraldehyde concentration was used.

Fig 3 SDS-PAGE and Coomassie brilliantblue staining results of ACCBP chemical crosslinking experiment.

5.Extracellular crystallization assay

Add an appropriate amount of ACCBP solution, water and MgCl₂ solution into the newly prepared saturated Ca(HCO₃)₂ to prepare 20μl solution containing 0, 8.0, 16.0, 40.0 mM MgCl₂. Drip the 20μl reaction system onto the cover glass. After 24 hours of reaction, observe the calcium carbonate crystals on the slides were studied by SEM.

Result: For better images, the calcium carbonate crystals were observed in different magnifications. It was obvious that when the concentration of Mg²⁺ was low, ACCBP induced the formation of typical square calcite crystals. When the concentration of Mg²⁺ reached 40mM, ACCBP induced the formation of more aragonite particles. Our results also indicated that the deletion of 16 N-terminal amino acids of ACCBP had no significant effect on the protein function.

Fig 4 The influence of Mg²⁺ to the crystallization results of ACCBP

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