Preparation method for competent host cell, and method for rapid and highly efficient transfer of exogenous substances into host cell and application thereof
A technology of host cells and exogenous substances, applied in the field of biological products and biotechnology applications, can solve the problems of high operation cost, large cell damage, low transformation efficiency, etc., and achieve the effects of simple operation, low damage and low cost
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Embodiment 1
[0041] Example 1. Using vacuum freeze-drying technology to quickly and efficiently transform exogenous substances into host cells and detect biochemical indicators
[0042] In this embodiment, the exogenous substance is taken as an example of the plasmid pET28b-Tat-EGFP, and the host cell is taken as Escherichia coli BL21(DE3) as an example. Reference as figure 1 As shown, the following operations are performed to transfer foreign substances into host cells:
[0043] 1) Purification, recovery and pretreatment of E. coli host cells
[0044] (1) Purify host cells: use LB solid medium (recipe: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar powder 15g / L) to culture and purify E. coli host cells BL21(DE3 ), to obtain Escherichia coli host cell monoclonal;
[0045] (2) Resuscitating host cells: Pick a single colony of E. coli host cells and inoculate them into 5 mL of LB liquid medium (recipe: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L), and recover overnight at 37°C...
Embodiment 2
[0064] Example 2. Using vacuum freeze-drying technology to prepare DH5α competent cells and rapidly transform pUC19
[0065] like Figure 5 As shown, the exogenous substance is the plasmid pUC19 (purchased from THERMO FISHER, 0.5 μg / μl) as an example, and the host cell is Escherichia coli DH5α as an example. The following steps are the same as in Example 1, and unless otherwise specified, basically the same expressions can be referred to for each other.
[0066] 1) Purification, recovery and pretreatment of E. coli host cells
[0067] (1) Purify host cells: use LB solid medium (recipe: tryptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar powder 15g / L) to culture and purify E. coli host cells (DH5α) by streaking at 37°C , to obtain Escherichia coli host cell DH5α monoclonal;
[0068] (2) Resuscitating host cells: pick DH5α monoclonal and inoculate them into 5 mL LB liquid medium (recipe: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L), recover overnight at 37°C and 220 rp...
Embodiment 3
[0077] Example 3. Preparation of Pichia pastoris GS115 Competent Cells by Vacuum Freeze-drying Technology and Rapid Transformation of pUC19
[0078] The exogenous substance is the plasmid pUC19 (purchased from THERMO FISHER, 0.5 μg / μl) as an example, and the host cell is Pichia pastoris GS115 as an example. The following steps are the same as those in Example 1 and Example 2, and unless otherwise specified, basically the same expressions may refer to each other.
[0079] 1) Purification, recovery and pretreatment of E. coli host cells
[0080] (1) Purify host cells: Use YPD solid medium (recipe: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agar powder 20g / L) to culture and purify Pichia pastoris GS115 by streaking at 30°C for two days, Obtain yeast cell monoclonal;
[0081] (2) Recovery of host cells: pick GS115 monoclonal and inoculate into 5mL YPD liquid medium (recipe: yeast extract 10g / L, peptone 20g / L, glucose 20g / L), recover overnight at 30°C, 220rpm (12-16 Hour...
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