Part:BBa_K1951004:Experience

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DesA production

Test of our biobrick proteins production using a SDS page and comassie

Production of the protein Des A has been tested into a PSB1C3 plasmide in a Escherichia Coli Tg1 strain.

In a LB lysine medium, strains have been cultivated from DO=0.2 (600nm)in 37°C room under 450rpm and induced between DO=0.4 and DO=0.6 with 0.2% arabinose.

After an over night culture, the well production of the protein has been shown by a SDS page and Comassie revelation. We tested the degradation of lysine into cadaverin by HPLC using a C18 column in a wild type strain and in a desA mutant strain.

DesA function demonstration

We had a look of the cadaverine production by the lysine decarboxylase DesA. Production has been detected by HPLC using C18 column after induction of the strain. Escherichia coli Tg1 strain was used as a wild type. Cadaverine production has been detected in this strain. In a Tg1 cadA mutant, cadaverine was also produced in a least quantity showing that an other pathway is responsible for the production of cadaverine. In the cadA mutant complemented by Bba_K1951004, the amount of cadaverine was recovered and even beyond the wild type production. Moreover, in the cadA mutant complemented by Bba_K1951011, the cadaverine level produced was even over the wild type and complemented Bba_K1951004 production

One the previous figure, cadaverine production has been mesured in different background strains. Cadaverine has been well detected in the WT. In the cadA mutant, the production of cadaverine is done even if the lysine decarboxylase is produced in a lesser quantity. We concluded that an other pathway can made cadaverine from lysine. When we have complemented the cadA mutant with our biobrick Bba_K1951004. Results have shown a recovery of the cadaverine biosynthesis and even beyond the rate obtained with the WT strain. Moreover, when we complemented the cadA mutant with the Biobrick containing the full operon desABCD, cadaverine catalysis was higher than in the WT strain. We explained this result because of the higher stability of this fragment. This experiment has shown that our Bba_1951004 is fonctionnal and allows the cadaverine production after induction.

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