Pseudomonas putida for preparing deoxidized violacein and uses thereof
A technology of Pseudomonas putida and deoxypurple, which is applied in the field of recombinant Pseudomonas putida, can solve the problems of difficult separation, low yield of deoxyviolet violacein, less research on properties and biological activities, and achieve simple separation and purification, Ease of industrial production and high yield
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Embodiment 1
[0025] Embodiment 1, the recombinant bacterium P.putida-Vio△D producing deoxyviolet violacein
[0026] 1) Gene cluster related to the synthesis of deoxyviolet bacterin
[0027] Insert Duganella sp. B2 CGMCC №2056 into liquid medium (starch 15g / L, ferrous sulfate 0.03g / L, potassium nitrate 1g / L, dipotassium hydrogen phosphate 0.7g / L, sulfuric acid Magnesium 0.5g / L, tryptophan 0.5g / L, pH 7.0), 25°C, 200rpm culture for 36 hours, according to the instructions of the Shanghai Sangon Genomic DNA Extraction Kit, the Genomic DNA of Duchenne B2 was extracted.
[0028] According to the sequence of the violacein gene cluster, 3 pairs of primers were designed by using 0ligo7.10 software. A part of the vioB and vioC genes was added, and the amplified product was named fragment B; P5 and P6 amplified the vioE gene, and the amplified product was named fragment C; there was a 48bp repeat sequence between the two primers P4 and P5 ( figure 1 ).
[0029] Table 1. PCR primer design
[0030] ...
example 2
[0046] Example 2, recombinant bacteria P.putida-Vio △ D production of deoxyviolet violacein
[0047] Insert P.putida-Vio△D into the E2 liquid medium (NaH 2 PO 4 2H 2 O1.3g / L, Na 2 HPO 4 12H 2 O3.0g / L, NH 4 Cl0.9g / L, K 2 HPO 4 ·3H 2 O7.5g / L, 100mM MgSO 4 ·7H 2 (010mL / L, glycerin 3mL / L, yeast extract 1.0g / L, pH is 7.0), the final concentration of tryptophan is 0.4g / L, 30 ℃, 200rpm shaking culture overnight. The next day, inoculate the inoculation amount of 10ml / 100ml into fresh E2 liquid medium containing kanamycin (50 μg / ml) and add tryptophan (final concentration 0.4g / L) at 30°C to continue the fermentation culture 3- 4h to OD 600 When it is 1.0, add the inducer n-hexane (0.05ml / 100ml) for induction, induce culture at 20°C for 30 hours, collect the bacteria by centrifugation, and extract the bacteria with ethanol to obtain a blue-purple substance.
[0048] Insert Duganella sp. B2 CGMCC №2056 into liquid medium (starch 15g / L, ferrous sulfate 0.03g / L, potassium nitr...
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