Method for increasing content of intracellular heme in escherichia coli
A technology of Escherichia coli and recombinant Escherichia coli, which is applied in the field of metabolic engineering and can solve problems such as increased cost and inconvenient operation
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Embodiment 1
[0035] According to SEQ ID NO.1, each 500bp homology arm of its upstream and downstream was designed, through the method of Red homologous recombination (see Datsenko KA.One-step inactivation of chromosome genes in Escherichiacoli K-12 using PCR products.Proceedings of the specific steps National Academy of Sciences of the United States of America, 2000,97(12):6640-6645)) knock out the mscS gene in Escherichia coli BL21, after sequencing verification, obtain the genetically engineered bacterium that knocks out the mscS gene, and name it for WT-M.
[0036] Inoculate the genetically engineered bacteria WT-M into LB medium, activate overnight at 37°C, 200r / min, transfer 500μL of a certain concentration of bacterial solution to 50mL of LB medium the next day, and incubate at 37°C, 200r / min Under culture, measure the bacterial concentration in real time, and make a growth curve, such as figure 2 shown.
[0037] Inoculate strain WT into LB medium, activate overnight at 37°C, 200r...
Embodiment 2
[0041] According to SEQ ID NO.2, the upstream and downstream 50bp homology arms were designed, and the aroG gene was knocked out on the basis of WT-M through the Red homologous recombination method. After sequencing verification, the genetic engineering of knocking out the aroG gene was obtained. bacterium, and named it as WT-MG, and obtained the genetically engineered bacterium.
[0042] Inoculate the genetically engineered bacteria WT-MG into LB medium, activate overnight at 37°C, 200r / min, and take 500μL of the activated bacterial solution the next day and transfer it to 50mL of LB medium, at 37°C, 200r / min Under culture, measure the bacterial concentration in real time, and make a growth curve, such as figure 2 As shown, there was no significant difference in the growth of genetically engineered bacteria WT-MG and WT.
[0043] After culturing for 7 hours, the hemoglobin content was detected, and the hemoglobin content in WT-MG was 2.86 μmol / L.
Embodiment 3
[0045] The glutamyl-tRNA reductase hemA gene (nucleotide sequence shown in SEQ ID NO.3) was connected to the pET28a vector through HindⅢ and EcoR I to obtain the recombinant plasmid pET28a-hemA, and then the recombinant plasmid was transferred into JM109 , spread the bacterial solution on an LB plate, culture at 37°C until a single clone grows, pick a single clone and perform sequencing verification, and the correct verification is a positive transformant. Inoculate the positive transformants into LB liquid medium, cultivate for 8-12 hours, extract the recombinant plasmid pET28a-hemA from the bacterial liquid, introduce the recombinant plasmid into the bacterial strain WT-M obtained in Example 1, and obtain the engineering bacterium pEA-M, The hemoglobin concentration was detected by fluorescence method, and the results showed that the hemoglobin content of pEA-M reached 45.8 μmol L -1 .
[0046] The pET28a plasmid was transformed into BL21 with no load, and the engineered ba...
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