Pichia pastoris expression medium preparation

2.1 LB (Luria-Bertani) medium:

Trypton l%

Yeast Extract 0.5%

NaCl l%

PH 7.0

Add 2% agar powder when making the plate. Autoclave at 121 Â° C for 20 minutes. Can be stored at room temperature. Zeocin 25ug / ml can be added when culturing pPICZÎ±A prokaryotic host strain TOP10F '.

2.2 LLB (Low Salt LB) medium:

Trypton l%

Yeast Extract 0.5%

NaCl 0.5%

PH 7.0

Add 2% agar powder when making the plate. Autoclave at 121 Â° C for 20 minutes. It can be stored at room temperature for several months. When used to cultivate pPICZÎ±A prokaryotic host strain TOP10F ', Zeocin 25ug / ml can be added and it can be stored at 4 â„ƒ for 1 to 2 weeks.

2.3 YPD (also known as YEPD)

Yeast Extract Peptone Dextrose Medium, (Yeast Extract Peptone Dextrose Medium, Yeast Extract Peptone Dextrose Medium, Dextrose Medium)

Trypton 2%

dextrose (glucose) 2%

+ agar 2%

+ Zeocin 100 Î¼g / ml

Liquid YPD medium can be stored at room temperature; agar YPD plates can be stored for several months at 4 â„ƒ. Add Zeocin 100ug / ml to become YPDZ medium, which can be stored at 4 â„ƒ for 1 to 2 weeks.

2.4 YPDS + Zeocin medium (Yeast Extract Peptone Dextrose Medium):

yeast extract 1%

peptone 2%

dextrose (glucose) 2%

sorbitol (Sorbitol) 1 M

+ agar 2%

+ Zeocin 100 Î¼g / ml

Regardless of whether it is liquid YPDS medium or YPDS + Zeocin medium, it must be stored at 4 Â° C for 1 to 2 weeks.

2.5 MGY

Minimal Glycerol Medium

(34% YNB; 1% glycerin; 4 * 10-5% biotin). Mix 800ml of sterilized water, 100ml of 10 * YNB mother liquor, 2ml of 500 * B mother liquor and 100ml of 10 * GY mother liquor, and store at 4 â„ƒ for 2 months.

2.6 MGYH

Minimal Glycerol Medium + Histidine (Minimal Glycerol Medium + 0.004% Histidine)

Add 10ml of 100 * H mother liquor to 1000ml of MGY medium, mix well, store at 4 â„ƒ, and the storage period is 2 months.

2.7 RD

Regeneration Dextrose Medium

(Contains: 1mol / L sorbitol; 2% glucose; 1.34% YNB; 4 * 10-5% biotin; 0.005% amino acid)

1. Dilute 186g of sorbitol to 700ml and autoclave;

2. After cooling, in a 45 â„ƒ water bath;

3. Mix 100ml of 10 * D, 100ml of 10 * YNB; 2ml of 500 * B; 10ml of 100 * AA and other mother liquors and 88ml of sterile water. mixing. Store at 4 â„ƒ.

2.8 RDH

Regeneration Dextrose Medium + Histidine (Glucose Regeneration Medium + 0.004% Histidine)

In the third step of RD medium preparation, 10 ml of 100 * H mother liquor is added, and the volume of sterile water is reduced to 78 ml at the same time, the rest of the preparation method is the same as RD. Store at 4 â„ƒ.

2.9 Preparation of RD and RDH plates

1. Make 186g of sorbitol and 15-20g of agar powder to 700ml, autoclave; after cooling, bath in 60 â„ƒ;

2. Refer to step 4 of RD / RDH liquid medium preparation, mix 100ml of 10 * D, 100ml of 10 * YNB; 2ml of 500 * B; 10ml of 100 * AA and other mother liquors, (10ml of 100 * H mother liquor) and 88 (78) ml of sterile water was mixed, preheated to 45 â„ƒ, mixed with the sorbitol / agar solution of step 1;

3. Prepare the plate quickly. It can be stored for several months at 4 â„ƒ.

2.10 Preparation of TOP agar for RD and RDH (commonly used for yeast coating)

1. Make 186g of sorbitol and 7.5 ~ 10g agar powder to 700ml, autoclave; after cooling, bath in 60 â„ƒ;

3. Place the TOP agar in a 45 Â° C water bath to cool, keep warm, and set aside.

2.11 MD and MDH

Minimal Dextrose Medium + (Histidine) Minimal Dextrose Medium + (0.004% Histidine)

(Contains: 1.34% YNB ;; 4 * 10-5% biotin; 2% glucose)

1. 100ml of 10 * YNB; 2ml of 500 * B and 100ml10 * D mother liquor, make up to 1000ml with 800ml of sterile water;

2. If MDH is prepared, add 10ml of 100 * H to the above MD;

3. If preparing a plate, add 15-20g of agar before sterilizing with sterile water. It can be stored for several months at 4 â„ƒ.

2.12 SOC medium:

Trypton l%

Yeast Extract 0.5%

NaCl 0.05%

Glucose (1mol / L) 2%

Autoclave at 121 â„ƒ for 20min, and store at 4 â„ƒ after cooling

2.13 MM medium:

M9 mother liquor: 64g disodium hydrogen phosphate, 15g potassium dihydrogen phosphate, 2.5g sodium chloride, 5.0g ammonium chloride plus 1L water sterilization

Take 200ml of M9 mother liquor, add 700ml of water, add 2ml of 1mol / l sterilized magnesium sulfate, add 100 microliters of sterilized 1mol / l calcium chloride (can be added or not). Note that the magnesium sulfate and M9 mother liquor should be sterilized separately or there will be precipitation. Adding 0.5% glucose on the basis of M9 medium is MM medium.

2.14 BMGY / BMMY medium:

Yeast powder: 1.0 g, peptone: 2.0 g, YNB: 1.34 g, 0.1 mol / L pH 6.0 (or pH 7.0) phosphate buffer, glycerin: 1.0 mL, add distilled water to 100 mL

2.15 BMM medium, full name: Buffered minimal methanol is the minimum buffered methanol medium, which is often used to express secreted proteins.

Preparation:

100mM pH6.0 potassium phosphate 1.34% YNB 4 Ã— 10-5% biotin 0.5% methanol

1. Sterilize 700ml water,

2. Cool to room temperature and add the following: 100ml 1M pH 6.0 potassium phosphate buffer, 100ml 10 Ã— YNB, 2ml 500 Ã— B, 100ml 10 Ã— M

3. Placed at 4 degrees, can be placed for 2 months

10 Ã— YNB (13.4% yeast nitrogen source base (YNB) contains ammonium sulfate but no amino acids)

Dissolve 134g of YNB in â€‹â€‹1000ml of water, filter and sterilize, heat until YNB is completely dissolved, and store at 4 degrees. Or use 34gYNB (without ammonium sulfate and no amino acids) and 100g ammonium sulfate for 1 year. Note that Pichia pastoris grows better at high concentrations of YNB.

YPD: the most basic culture; BMGY: culture before induction expression; BMMY: induction expression; MD: his + screening after electrotransformation.

YEPD cannot replace BMGY, because there is glucose, so the residual glucose will affect the next induced expression. However, there is a method that is feasible, that is, to replace with YPG medium, just replace the glucose in YEPD with 3% glycerol, which can also reduce costs. After all, the shake flask cannot be compared with the fermentor, and the residual glycerin will inhibit the use of methanol.

Methanol, potassium phosphate and biotin can only be added after BMGY and BMMY are sterilized. When preparing BMMY, it is not necessary to use 5% filtered sterilized methanol. After sterilization, add 100% methanol to the concentration you want.

YNB can be autoclaved, no problem, or 0.22um filtration treatment, aspartic acid and threonine should be added after the medium is autoclaved; when YPD is equipped, YPD can be added together for sterilization, but the time cannot be too long , The temperature can not be too high, generally 121-125 degrees 12-15 minutes is enough. If the time is too long and the temperature is too high, it may cause YPD coking. Glucose and nitrogen-containing compounds together are prone to Maillard reaction, which is a taboo in formulated media. If the color is very dark, it can't be used. Or the medium containing glucose and / or YNB is autoclaved at 108 degrees for 35 minutes.

The small amount of fermentation can actually remove the YNB and biotin in the medium. The medium is cheap, easy to operate, and can be directly sterilized. The effect is also very good (the effect is not worse than that containing YNB).

If you use your own configured medium, such as corn extract, malt extract, wheat bran extract, etc., you can use the supplement to maintain the nutritional needs of the culture medium without changing the medium.

Large-scale fermentation with inorganic salts saves money.

In large-scale fermentation, the acceleration of methanol flow should not increase too fast. In addition, the use of pure oxygen is not expensive, buy a steel bottle in Wuhan about 600 yuan, a bottle of pure oxygen 20 yuan. It is estimated that 3-4 bottles of oxygen are in a batch about 100h.

About the choice of Tryptone and Peptone:

1) Tryptone cultured E.coli instead of Peptone can be used for some yeasts, such as general expression yeast. However, it is absolutely not acceptable for yeasts. For example, two-hybrid AH109, â€‹â€‹Y187 and other expression yeasts are not good enough .

2) Although Tryptone and Peptone are both nutritional peptones, the nutritional components are different. Even the general expression yeast can grow in Tryptone, the growth state is not as good as in Peptone. But tryptone and peptone are a little different in content, nothing else The difference is that the purpose of using peptone is not to provide a nitrogen source. The nitrogen source is the YNB in â€‹â€‹the medium.

3) If the requirements are not high, the general use is also OK. Use Peptone, difco, Jingmei agent, and even other domestic peptones to cultivate most yeasts as normal as possible.

4) The medium containing Peptone is very dark, and the color should be removed when purifying the expressed protein, so troublesome follow-up treatment is also required. After using Tryptone, the color of the medium becomes very light, which is similar to that of LB (liquid), and the subsequent treatment is simpler. The invitrogen manual says that the role of peptone is to prevent the target protein from being hydrolyzed by some enzymes. The purpose of adding peptone is to competitively inhibit the hydrolysis of the target protein, because peptone is a small short peptide that is a substrate for some enzymes.

Cultivating Pichia pastoris, the book says that the BIOTIN stock solution is an aqueous solution, but in fact BIOTIN is difficult to dissolve in water and can be heated in a water bath.

There are 3 options for preparing 500 Ã— BIOTIN stock solution (0.02%):

1). The lazy person dissolves Biotin directly in deionized water, and after overnight, it can basically be dissolved;

2), the acute matter is to make the solution into 0.02N NaOH, it is easy to dissolve;

3). Heating in water bath, the temperature cannot be higher than 50 degrees.

D-Biotin is a bioactive biotin, which is vitaminH. In the metabolism of Pichia pastoris, it acts as a prosthetic group for various enzymes. Natural medium can not be added separately, because YNB, yeast powder and peptone all contain a certain amount of biotin, but it must be added for high-density fermentation. MD and MM belong to the basic medium, and no component will contain trace growth factors (such as biotin). So it must be added.

Attachment: inorganic medium formula

BSM inorganic medium:

85% H3PO4 26.7ml / L

CaSO4? 2H2O 0.93g / L

K2SO4 18.2g / L

MgSO4? 2H2O 14.9g / L

KOH 4.13g / L

Glycerin 40g / L

PMT1 4.0ml / L

100% ammonia water to adjust pH5.0 (1 bottle of 50ml plus 800ul ammonia water) or 10g / L ammonium sulfate to adjust pH5.0, ammonia water is used to adjust pH on the tank (cheap and convenient). Adjust pH 6.0 before inducing expression. pH5.0 is to prevent BSM from forming calcium phosphate precipitation, and pH6.0 is the optimal pH for expressing HSA fusion protein.

After autoclaving BSM, add 4.0ml / L of filter-sterilized PTM1 (trace element) 4.0ml / L. 100% 500ml methanol plus 6ml PMT1 is used to add methanol to induce expression of the fusion protein of interest.

PMT1 (1L) formula:

CuSO4? 5H2O 6.0g / L

KI 0.088g / L

MnSO4? H2O 3.0g / L

Na2MoO4? 2H2O 0.2g / L

H3BO3 0.02g / L

CoCl2? 6H2O 0.5g / L

ZnCl2 20.0g / L

FeSO4? 7H2O 65.0g / L

Biotin 0.2g / L

Concentrated H2SO4 5.0ml

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