Overview of Experiments
1. Production Strains
In order to ensure that the bacteria we are working with are really the ones we want to work with, a 16S rDNA PCR was performed.
As an antibiotic resistance will be used in our later experiments to select transformants, we checked for any native antibiotic resistance against ampicillin, kanamycin, tetracyclin and chloramphenicol in an LB agar plate growth test. It turned out that kanamycin was the only antibiotic, against which none of our strains showed any resistance. For this reason, we decided to use only vectors with kanamycin resistance for our later projects.
After this, we prepared electro competent cells from our strains. All of our strains were grown on LB medium.
2. BioBricks
Our BioBricks were designed using the software SnapGene. The original Genes for our B12 binding proteins, and the torA signal sequence were codon optimized for E. coli.
The DNA containing the Genes were synthesized and friendly provided by IDT Integrated DNA Technologies.
In case of MutB, the length of the gene exceeded the maximum length of 2000 bp from IDT. For this reason, we designed two DNA parts, with a natural occurring HindIII restriction site at both ends. We intended to fuse these both genes by ligation. However, after having diffuculties to fuse the genes by ligation, we decided to fuse them by fusion PCR. In order to multiply the DNA for or cloning experiments, and to equip our genes with restriction sites, PCR was used.
For our purpose, we were searching for a vector with (a) kanamycin resistance and (b) araBAD operon. We thus decided to use the pBAD202 expression system. This vector, however, is equipped with a thioredoxin fusion protein which increases the rate of expression, but prohibits the physiological function of our protein. For this reason, we designed primers for an autarkic synporter protein. In order to test the dependence of the expression system on the B12 export rate, we also decided to use different other vectors.
For vectors ligation, we used a standart ligase reaction, for the pBAD202 expression, which works without ligation but topoisomerase reaction, we followed the manufacturer's protocol.
3. Transformation and Cultivation
For our transformation experiments, we used electroporation for all our self prepared competent cells. For the heat competent E. coli Top10 cells provided with the pBAD202 expression system, we used the manufacturer's heat shock transformation protocol.
For vector multiplication, E. coli cells (Top10 for pBAD202 plasmids, DH5alpha for all other plasmids) were transformed with the constructed plasmids. After the transformation, the cells were cultivated on LB Medium. Transformants were selected, and the respective clones were picked, cultivated in LB medium, and plasmids were extracted.
The Plasmids were then introduced into the production strains: Raoultella planticola ATCC 33531, Shimwellia blattae ATCC 29907, and Salmonella typhimurium TA100.
For the expression experiments, the all cells were cultivated and the expression induced in RM medium under aerobic conditions. As S. blattae is known to produce Vitamin B12 only under anaerobic conditions, the expression was induced under anerobic conditions in RM medium.
The induction was performed using different concentrations of l-arabinose and different induction time spans.
4. Expression Test
To test for the expression of our synporter protein, the cell lysates of the expression cultures we submitted to an SDS-PAGE. From the SDS Gels, Western Blots were performed using anti-Poly-Histidine-tag antibodies.
5. Vitamin B12 Assay
To test the physiological function of our synporter protein, different assays for B12 detection were performed. For the assays, (a) the whole cell filtered medium, and (b) the periplasm fraction of the cells were examined.
The periplasm fraction of the producing cells was gained by ultrazentrifugation.
5.1. Microbial Assay
The microbial B12 assay relies on the growth diameter of the E. coli ATCC 10799 strain on an agar plate, in dependence of the Vitamin B12 (or methionine) concentration in the medium.
For this purpose, agar plates with synthetic Guttman medium (free of B12 and amino acids, 1.5% agarose) were prepared. The E coli detection cells were grown in liquid synthetic Guttman medium supplemented with selected amino acids (no methionine) and Vitmamin B12. Afterwards, the cells were washed with PBS buffer three times, in order to make the cells virtually free of Vitamin B12. The cells were resuspended in PBS buffer, and mixed with warm, liquid Guttman medium (free of B12 and amino acids, 0.8% agarose), and then poured on the prepared agar plates. For the assay, small paper platelets (usually used for antibiotic tests) were placed on the top agar layer, and soaked with differently concentrated calibration solutions of Vitamin B12. The plates were incubated overnight, and the diameters measured after 16 h. From the different diameters, dependent of the Vitamin B12 concentration, a calibration curve was calculated.
In order to determine the B12 content of our medium and periplasm fraction, the assay plates were prepared exactly as described above, and paper platelets were soaked with the respective solution. Comparing to the calibration curve, the diameter indicates the B12 amount.
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