Cloning
For this part, our aim was to clone our synthesized genes into a vector plasmids, and introduce them in our chassis organisms. Our control experiments show that we succeeded with cloning our constructs into the pBAD202 plasmid, and introducing them into our chassis organisms S. blattae and R. planticola.
Fig. 1: Control restriction digest. The Gel shows different sized fragments after the restriction of the assembled plasmids. According to the information given in Tab. 1, the correct plasmid cloning worked in the clones BtuF 4, GlmS 4 and GlmS 5.
Tab. 1: Sizes of the expected gene fragments glmS btuF after restriction digest of the assembled pBAD202 plasmid.
| Binding Domain |
btuF |
glmS |
| Fragment Size |
927 bp |
540 bp |
| Size in pBAD202 (4448 bp) |
5375 bp |
4988 bp |
| SmaI Digest in pBAD202 |
Correct: 386 bp + 4989 bp
Only cut once w/o insert 4448 bp
Incorrect: 905 bp + 4470 bp |
No SmaI restriction site |
| AccI Digest in pBAD202 |
|
Correct: 2578 bp + 2410 bp
Incorrect: 2996 bp + 1992 bp
Only cut once w/o insert 4448 bp |
Fig. 3: Transformation control. The agarose gel of the restriction digested vectors shows that S. blattae and R. planticola were successfully transformed with the desired plasmid DNA.
Expression
In the next major step, we wanted to prove that our engineered Synporter protein was expressed in our chassis organisms. Moreover, we wanted to prove that our Synporter protein is exported into the periplasm.
Our general induction experiments showed that the genes of both of our his-tagged Synporter proteins TorA-BtuF and TorA-GlmS were expressed successfully in R. planticola and S. blattae. Examining the fractions of our cells (periplasm, speroplasts, and cytoplasm), our Synporter proteins were detected outside of the cytoplasm.
Our TorA-BtuF Synporter protein has a calculated molecular weight of 37.22 kDa and contains a polyhistidine-tag, so we expected a protein band on the western blot in the area of the 35 kDa mark, which was detected. Our TorA-GlmS Synporter protein has a calculated molecular weight of 22.6 kDa and contains a polyhistidine-tag, so we expected a protein band on the western blot in the area obetween the 25 kDa mark and the 15 kDa mark, which was also detected.
Fig. 4: Western blot with different cellular fractions. Western Blot analysis was performed with the periplasma fraction, and the spheroplast fraction and the disrupted periplasma as control.
Function
Finally, we wanted to prove that the counterpart of our Synporter proteins, the Vitamin B12, was also exported into the periplasm. Therefore, we let our organisms produce Vitamin B12, fractionated our cells as described above, and performed a photometric B12 assay. Compared to empty plasmid controls, we could detect significantly higher amounts of Vitamin B12 in the cytoplasm, as also in the periplasm, when the synporter proteins were expressed (Fig. 5). This increased production is mainly due to the fact that the cells have to compensate for the B12 leaving the cytoplasm bound to the Synporter.
Fig. 5: Final results of the photometric assay. The plot shows the quantity of B12 per amount of protein, in the periplasmic fraction and the cytoplasmic fraction.
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