Mutated mycobacterium smegmatis capable of secreting niacin and construction method of mutated mycobacterium smegmatis
A technology of Mycobacterium smegmatis and a construction method is applied in the field of bioengineering to achieve the effects of low cost, easy amplification and simple production conditions
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Embodiment 1
[0032] Example 1: Construction of plasmid pMHS-NrtR ms
[0033] 1) Take Mycobacterium smegmatis mc 2 155 genomic DNAs were used as templates, and the primer pairs shown in SEQ ID No. 1-4 were used for PCR amplification. like figure 1 As shown, the PCR product nucleic acid gel electrophoresis was used to separate the target DNA fragments, and the Omega company gel back kit was used to recover the target DNA fragments shown in SEQ ID No. 5 and 6. The recovered DNA was digested with Van91I restriction endonuclease, and the digested DNA was recovered by Omega's Cycle-pure recovery kit for use;
[0034] 2) Escherichia coli DH5α strain containing the pMHS plasmid was inoculated with LB liquid medium (adding 150 μg / mL hygromycin B) and cultured overnight at 37°C, and then the bacteria were collected to extract the plasmid using the Omega plasmid extraction kit to obtain the pMHS plasmid Carry out nucleic acid gel electrophoresis after Van91I restriction endonuclease digestion and...
Embodiment 2
[0037] Example 2: Construction of plasmid pHAGE-NrtR ms
[0038] 1) LB were cultured with pHAGE (amphicillin resistance) and pMHS-NrtR ms (Hygromycin B resistance) Escherichia coli DH5α strain of the plasmid and use the plasmid extraction kit of Omega company to extract the plasmid, and the two obtained plasmids are linearized with PacI restriction endonuclease and then reacted with T4DNA ligase at 16°C overnight. The ligation product was packaged and transformed into Escherichia coli HB101 competent cells using a packaging kit (EPICENTREBiotechnologies: MaxPlax Lambda Packaging Extracts), and then coated on an LB solid plate (containing 150 μg / mL hygromycin B), and the plate was placed in a 37°C constant temperature incubator After culturing overnight, single clones grown on the plate were picked, inoculated with LB liquid medium (containing 150 μg / mL hygromycin B) and cultured overnight on a shaker at 200 rpm at 37°C, and the plasmid was extracted using Omega’s plasmid ext...
Embodiment 3
[0039] Embodiment 3: Preparation of recombinant TM4 phage
[0040] 1) Preparation of Mycobacterium smegmatis mc 2 155 competent cells: Mycobacterium smegmatis mc 2 155 was inoculated in 5ml 7H9 liquid medium, cultured at 37°C with shaking at 200rpm until the logarithmic growth phase (OD0.5-1.0); the culture was inoculated in fresh 100mL 7H9 liquid medium at a ratio of 1:100, overnight at 37°C Cultivate until the OD600 reaches about 0.6, collect the bacteria by centrifugation at 5000rpm at 4°C for 10 minutes, wash the bacteria with pre-cooled 10% sterile glycerol at least twice, and finally add 10 mL (appropriate amount) of pre-cooled 10% glycerol, weigh After suspending the bacteria, freeze them in separate devices at -80°C for later use;
[0041] 2) will construct the correct positive plasmid pHAGE-NrtR ms Add DNA to 200 μL of M. smegmatis mc 2 155 electroporation-competent cells were incubated on ice for 10 minutes, then transferred to a 2mm BTX electroporation cup, wip...
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