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For the proof of our concept we showed that our modified Clostridium strain is able to grow and thereby expresses the acetyl-CoA (acsB) gene added with a plasmid. Therefore, many repetitive steps of measuring bacterial growth, PCR´s and sequencing was required.
To prove our concept of adding foreign genes to a bacterium strain we performed lab work. The first step was to verify that the plasmid we wanted to insert into the bacterium Clostridium saccaroperbutylacetonicum contained the acetyl-CoA synthetase (acsB1) gene, which was earlier extracted from Acetobacterium woodii. Therefore, a gel electrophoresis was performed and the occurring bands compared. The results indicated that the gene was successfully inserted onto the plasmid. In order to ensure that the visible band was acsB1 and not another fragment with the same length we sequenced our plasmid and it proved to be correct.
In the next step we transformed our plasmid into C. saccharoperbutylacetonicum. Growing colonies of our modified Clostridium stain were analyzed for the occurrence of the acsB1 gene. Again, gel electrophoresis was performed to get a first inside if the clones had possibly inherited the gene. With further sequencing the appearance of the plasmid and acsB1 was verified. Since we had, at this point, proofed that acsB1 was successfully added into C. saccharoperbutylacetonicum we finally needed to ensure that it was also expressed. Therefore, we performed a qPCR which results demonstrated that the acsB1 gene is present and expressed in our organism. These results show that our modified strain is able to generate acetyl-CoA to complement the Wood-Ljungdahl pathway.
Additionally, growth curves were conducted to display that our modified Clostridium strain was able to grow and exhibit no drawbacks from our plasmid.