CN101050420A - Breeding medium for preparing cells in competence of electric transform in high effectively, and application - Google Patents

Abstract

This invention relates to a YENB culture medium for preparing high-efficiency electroporation-competent cells. The YENB culture medium comprises (per liter): yeast extract for cell culture 6-9 g, nutrient broth for cell culture 6-9 g, and water. The YENB culture medium can prepare high-efficiency electroporation-competent cells by controlling cell primary and secondary growth time, centrifugal speed and time. The conversion rate is 2.19X1010 cfu/mu.g pUC19 DNA, which is higher than imported culture medium, while the cost is only 5% that of imported culture medium. The culture medium lays a basis for high-efficiency conversion of large fragment DNA genomic library.

Description

A kind of substratum and application thereof for preparing efficient electric transformed competence colibacillus cell

Technical field

The present invention relates to electric transformed competence colibacillus cell, specifically a kind of substratum and application thereof for preparing competent cell, use this special culture medium culturing Gram-negative bacteria---(Escherichia coli, E.coli) the DH10B bacterial strain prepares efficient electric transformed competence colibacillus cell to intestinal bacteria.

Background technology

Conversion is the gordian technique that in the molecular cloning foreign DNA is imported recipient cell.Find to use CaCl from Mandle in 1970 and Higa ₂The cell of handling can be by after the lambda bacteriophage dna transfection, and 1972 and people such as Cohen in 1973 use the same method first successfully with the R factor and recombinant plasmid dna importing intestinal bacteria, thereby have established the genetic transformation technology.The method that transforms is a lot, experiment demand as Calcium Chloride Method, Hanahan method and Inoue method etc. can satisfy, but these method transformation efficiencies are lower, can reach 10 ⁷～10 ⁸Cfu/ μ g plasmid DNA, and complicated operation, repeatability is not high.And set up big fragment gene group library, as the BAC library, then need higher with transformation efficiency, the electric method for transformation that the result is more stable (Taketo A.DNA transfection of Escherichia coli byelectroporation.Biochim Biophys Acta, 1988,949 (3): 318-324).

The ultimate principle that electricity transforms is to utilize the momentary impulse electric field action in recipient cell, makes cell membrane component polarized, and produces potential difference on the cytolemma both sides.When potential difference surpasses a certain critical level, the cytolemma part is breakdown, forms some reversible holes, and pore size is enough to allow macromole and small molecules enter or discharge from cell.

Dissimilar substratum has certain influence to the transformation efficiency of competent cell.The substratum nutritive ingredient is high more, and the growth conditions of cell is just good more, and transformation efficiency is just high more; Ionic strength is more little, to the negative impact that transforms also more little (Dower W J just, Miller J F, Ragsdale C W.Highefficiency transformation of E.coli by high voltage electroporation.NucleicAcids Res, 1988,16 (13): 6127-6145).Because in the electroporation system, low ionic strength is to keep the key of certain hour high field intensity.The electrolyte concentration of electric shock in the liquid is high more, easy more recipient cell is formed invalid discharge or bypass discharge, can not produce the transmission duct.In addition, the strong current system temperature that can cause shocking by electricity raises the bacterium mass mortality suddenly moment.

The growth period of competent cell is one of important factor of the electric transformation efficiency of influence.Be difficult to realize efficient the conversion with being in the logarithmic growth early stage or the competence of the cell preparation in latter stage.Because the different in kind of cell, the kind difference of used substratum, the growth time of correspondence just had very big difference when bacterium reached the highest transformation efficiency.

Efficient electric transformed competence colibacillus cell is the assurance that makes up the especially big fragment gene group of high quality genomic library library.Many external well-known biological reagents such as Invitrogen, Stratagene, Lucigen company all has high-quality electric transformed competence colibacillus cell to sell, but costs an arm and a leg.With InvitrogenElectroMAX DH10B Competent Cell is example, though its transformation efficiency can reach 1 * 10 ¹⁰Cfu/ μ g pUC19 DNA, but per 500 μ l price Gao Da $258 at 150kb, contain 150000 clones' BAC library for an average fragment of inserting, and only one of competent cell will spend Jin $30000.

Summary of the invention

The object of the present invention is to provide the substratum and the application thereof of the preparation competent cell that a kind of cost is low, effect is good.

To achieve these goals, technical scheme of the present invention is as follows:

A kind of substratum for preparing competent cell contains in every liter of YENB substratum, is used for the yeast extract 6～9g of cell cultures, is used for the nutrient broth 6～9g of cell cultures, and surplus is a water.

Described YENB substratum can be used for cultivating Gram-negative bacteria---coli strain, prepares efficient electric transformed competence colibacillus cell.

Described bacterial strain is an intestinal bacteria DH10B bacterial strain, and the condition of once cultivating the DH10B competent cell is: single colony inoculation 8～12ml YENB cultivates based on 50ml point end plastic centrifuge tube, 37 ℃, the following 9～11h that cultivates of 240～260rpm; The condition of second incubation DH10B competent cell is: the DH10B cell that will once cultivate is according to 1: 1000 ratio inoculation YENB substratum, and 37 ℃, 180～220rpm are cultured to OD ₅₅₀Collected bacterium at=0.78～0.80 o'clock.

The present invention has following advantage:

1, preparation is simple, cost is low.YENB is that a kind of application does not have salt culture medium very widely.The used most of reagent of preparation competent cell is homemade analytical pure, as substratum, glycerine etc., substituted expensive import reagent by homemade analytical pure, thereby greatly reduce material cost, make that the material cost of preparation competent cell only is about 5% of a commodity competent cell price.

2. it is convenient to use.SOB substratum (no Mg ²⁺) saltiness nearly 0.7 ‰, and YENB is a kind of no salt culture medium, during therefore with its preparation competent cell, is not very strict to the requirement of washing, only need 1～2 washing just bacterial metabolism impurity and trace salt ion remaval can be greatly reduced the possibility of invalid discharge and bypass discharge.The YENB substratum is widely used in the preparation of various E.coli electricity transformed competence colibacillus cells and cultivates, and has ubiquity.

3. nutritious, can satisfy the growth needs of most of cell.YENB is not only a kind of no salt culture medium (salt-free media), and nutrient broth wherein (Nutrient Broth) nutritive ingredient is abundant, can satisfy the growth needs of most of cell, good with the DH10B competent cell growth conditions of YENB medium preparation.Therefore the YENB substratum can be widely used in the preparation process of most microbial culture and competent cell.

4. result of use is good.Competent cell growth conditions and transformation efficiency with the present invention's preparation obviously are better than preparing traditionally the used SOB substratum of electric transformed competence colibacillus cell; Comparing with the SOB substratum, owing to there is not the existence of salt ion, is not very strict to the requirement of washing, because washing times obviously reduces, also just little many to the damage of competent cell, so the transformation efficiency of competent cell obviously raises than SOB substratum, can reach 2.19 * 10 ¹⁰Cfu/ μ g pUC19DNA; Surpassed transformation efficiency with kind commodity competent cell (Invitrogen ElectroMAX DH10B Competent Cell), and can be in the medium-term and long-term preservation of cryogenic refrigerator.This technology provides technical foundation for the efficient conversion in the big fragment gene group of genomic library especially DNA library.

Description of drawings

Fig. 1 is the influences of different incubation times to prepared fresh competent cell transformation efficiency.

Embodiment

Novel culture medium YENB is as the substratum of preparation DH10B competent cell, be a kind of by yeast extract ( YEast EXtract) and nutrient broth ( NUtrient BRoth) the no salt culture medium that mixes;

YENB culture medium prescription and preparation method:

Prepare every liter of substratum, in 950ml ultrapure water (Millpore product), add: yeast extract 6～9g (the article No. LP0021 of OXOID company that is used for cell cultures; Worker's article No. is given birth in Shanghai: BN5245 etc.), be used for nutrient broth 6～9g (Gibco company article No.: 0003-17 of cell cultures; Tianjin Ao Jia Science and Technology Ltd. article No.: 05443 etc.), shake container and dissolve fully until solute.Regulate pH to 7.0 with 5mol/L NaOH, be settled to 1L with ultrapure water.At 15psi (1.05kg/cm ²) steam sterilizing 20min under the high pressure.

Embodiment

1.YENB the substratum preparation: every liter of substratum adds in the 950ml ultrapure water: yeast extract 8g, nutrient broth 8g shakes container and dissolves fully until solute.Regulate pH to 7.0 with 5mol/L NaOH, be settled to 1L with ultrapure water.At 15psi (1.05kg/cm ²) steam sterilizing 20min under the high pressure.

2. substratum is preferred:

Adopt nutritive ingredient YENB, SOB (the no Mg different with ionic strength ²⁺), 2 * YT, LB substratum carry out the selection optimization (SOB, 2 * YT, LB prescription are seen " molecular cloning " third edition) of substratum;

The culture condition of DH10B competent cell is as follows: take out the DH10B bacterial classification from Ultralow Temperature Freezer and rule at nonresistant LB solid medium flat board, be inverted for 37 ℃ and cultivate 14～16h.The individual mellow and full glossiness single colony inoculation 10ml YENB of picking cultivates based on 50ml point end plastic centrifuge tube, and 37 ℃, 250rpm are cultivated 10h down.With 1: 1000 ratio inoculation non-resistant YENB liquid nutrient medium (as, 500 μ l culturing cells inoculation 500ml YENB substratum), 37 ℃, 200rpm are cultured to suitable OD with a subculture ₅₅₀, take out culture and ice bath 20min immediately.

Utilize YENB, SOB (no Mg ²⁺), the fresh competent cell (OD of 2 * YT, LB medium preparation ₅₅₀=0.78) transforms.Experiment condition is 1 μ l pUC19 DNA (2pg/ μ l), 40 μ l competent cells, pulse field intensity 21kV/cm.The result is as shown in table 1, and the average transformation efficiency of the fresh competent cell of usefulness YENB medium preparation is the highest, reaches 2.19 * 10 ¹⁰Cfu/ μ g pUC19DNA.

Table 1, different sorts substratum are to the influence of competent cell transformation efficiency

	YENB	SOB	2×YT	LB
					21kV/cm	2.19×10 10	1.12×10 10	8.24×10 9	2.56×10 9

The transformation efficiency calculation formula is seen " electricity of competent cell transforms and efficiency test " part, transformation efficiency unit: cfu/ μ g pUC19 DNA.

3, the optimization of competent cell second incubation optimum growh time:

With YENB is the culture medium culturing cell, collects OD respectively ₅₅₀=0.30,0.40,0.50,0.60,0.70,0.80,0.90,1.00,1.10 o'clock bacterium liquid prepares fresh competent cell and transforms.Conversion condition is the same.The result adopts mid-log phase OD as shown in Figure 1 ₅₅₀The competence transformation efficiency of=0.78～0.80 cell preparation is the highest.

The top condition of once cultivating the DH10B competent cell is: single colony inoculation 10ml YENB cultivates based on 50ml point end plastic centrifuge tube, 37 ℃, the following 10h that cultivates of 250rpm;

The optimal growth condition of second incubation DH10B competent cell and growth time are: the DH10B cell 500 μ l that will once cultivate inoculation 500ml YENB cultivates based on 2500ml glass triangle bottle, and 37 ℃, 200rpm are cultured to OD ₅₅₀Collected bacterium at=0.78～0.80 o'clock, the competent cell transformation efficiency for preparing under this condition reaches 2.19 * 10 ¹⁰Cfu/ μ g pUC19 DNA.

4, the preparation of competent cell and frozen ^*

Plant and instrument: Millipore company pure water system; The cabinet type refrigerated centrifuge of Sorvall GSA; 37 ℃ of constant incubators and shaking table.

(1) takes out the DH10B bacterial classification from Ultralow Temperature Freezer and rule, be inverted for 37 ℃ and cultivate 14～16h at nonresistant LB solid medium flat board;

(2) the individual mellow and full glossiness single colony inoculation 10ml YENB of picking cultivates based on 50ml point end plastic centrifuge tube, and 37 ℃, 250rpm are cultivated 10h;

(3) 500 μ l cultures are added 500ml YENB substratum (ratio inoculation in 1: 1000), 37 ℃, 200rpm are cultured to OD ₅₅₀=0.78～0.80 o'clock, ice bath 20min immediately, during rock triangular flask gently 2～3 times;

(4) culture is changed in the 500ml centrifugal bottle of precooling, Sorvall GSA 3000 rotors, 0～2 ℃, the centrifugal 10～15min of 4000rpm carefully abandon supernatant;

(5) the ultrapure water re-suspended cell of isopyknic precooling, 0～2 ℃ of centrifugal 10～15min of 4000rpm carefully abandons supernatant;

(6) 10% glycerine re-suspended cell of isopyknic precooling, 0～2 ℃ of centrifugal 10～15min of 5000rpm carefully abandons supernatant ^*

(7) every liter of culture is resuspended with 2ml 10% precooling glycerine, and liquid nitrogen flash freezer 3min puts into the cryogenic refrigerator preservation after every pipe 40 μ l packing.

^*Employed plant and instrument and experimental article must guarantee totally pollution-free, and the entire operation process should be carried out in low temperature environment as far as possible.

^*Very loose behind the cell centrifugation of 10% glycerine washing, carefully take out centrifugal bottle, want significant care when toppling over supernatant.Inevitable loss small amounts of cells in this process, being sure not blindly to improve rotating speed increases competent cell output, because along with the increase of rotating speed, the damage of pair cell is more serious, thereby has reduced transformation efficiency;

5, the electricity of competent cell transforms and efficiency test

Plant and instrument: the Micropulser electroporation apparatus that Bio-Rad company gives birth to; 37 ℃ of constant incubators and shaking table.

(1) gets 1 μ l pUC19 DNA (2pg/ μ l) and mix the precooling pole cup that the back adds 1mmgap with 40 μ l competent cells, avoid producing bubble, pulsed field intensity is 21kV/cm, and the electric shock back adds 960 μ l normal temperature non-resistant SOC substratum rapidly, and 37 ℃ of 200rpm cultivate 1h.Each condition repeats 6 times, and sets up blank negative control;

(2) get 1 μ l, 2 μ l, 5 μ l respectively, 10 μ l bacterium liquid points are containing on the LB flat board of appropriate amounts of ammonia penicillin G, are inverted for 37 ℃ and cultivate 12～14h, counting also calculates transformation efficiency according to following formula.

$\frac{\mathrm{CFU\; on\; plate}}{\mathrm{pg\; pUC}19\mathrm{DNA}}\×\frac{1\×{10}^6\mathrm{pg}}{\mathrm{\μg}}\×\frac{\mathrm{volume\; of\; transformants}}{\mathrm{volume\; plated}}\×\mathrm{dilution\; factor}$

The present invention is by once cultivating the optimization with the second incubation condition to competent cell, and the fresh competence transformation efficient that compares YENB, SOB, 2 * YT, four kinds of different medium preparation of LB, finally determined the optimal culture condition of DH10B competent cell and optimized the substratum of YENB as the efficient DH10B competent cell of preparation.Utilize the YENB substratum, successfully prepared efficient electric transformed competence colibacillus cell by important parameters such as control cell growth time, rotating speed when centrifugal and times.

Claims (5)

1. substratum for preparing competent cell, it is characterized in that: contain in every liter of YENB substratum, be used for the yeast extract 6～9g of cell cultures, be used for the nutrient broth 6～9g of cell cultures, surplus is a water.

2. according to the described substratum of claim 1, it is characterized in that: contain in every liter of YENB substratum, be used for the yeast extract 8g of cell cultures, be used for the nutrient broth 8g of cell cultures, surplus is a water.

3. the application of the described preparation competent cell of claim 1 substratum is characterized in that: the described YENB substratum of claim 1 is used to cultivate Gram-negative bacteria---coli strain, prepares efficient electric transformed competence colibacillus cell.

4. according to the application of the described preparation competent cell of claim 3 substratum, it is characterized in that: described bacterial strain is an intestinal bacteria DH10B bacterial strain, the condition of once cultivating the DH10B competent cell is: single colony inoculation 8～12ml YENB cultivates based on 50ml point end plastic centrifuge tube, 37 ℃, the following 9～11h that cultivates of 240～260rpm.

5. according to the application of the described preparation competent cell of claim 4 substratum, it is characterized in that: described bacterial strain is an intestinal bacteria DH10B bacterial strain, the condition of second incubation DH10B competent cell is: the DH10B cell that will once cultivate is according to 1: 1000 ratio inoculation YENB substratum, and 37 ℃, 180～220rpm are cultured to OD ₅₅₀Collected bacterium at=0.78～0.80 o'clock.

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