產品介紹

基 因 型Δ(ara-leu)7697ΔlacX74phoAPvuIIphoRaraD139ahpCgalEgalKrpsL(DE3)F[lac+lacIqpro]gor522::Tn10 trxB pLysSRARE2 (CamR , StrR , TetR )簡 要 說 明Rosetta-gami(DE3)pLysS 菌株聚合了不同原核表達菌株的優勢：?Rosetta-gami賦予其 Rosetta和Origami的優點——補充大腸桿菌缺乏的6 種稀有密碼子(AUA， AGG， AGA， CUA，CCC， GGA) 對應的tRNA，提高外源基因的表達水平，并且包含突變的硫氧還蛋白還原酶（thioredoxin reductase）(trxB) 和谷胱甘肽還原酶(glutathione reductase)(gor)基因，表達主要還原途徑的兩個關鍵酶。有利于形成正確折疊的含有二硫鍵的蛋白，增強蛋白的可溶性。?該菌株染色體整合了λ噬菌體DE3區（DE3區含有T7噬菌體RNA聚合酶），適合T7啟動子誘導的蛋白表達。?該菌株攜帶的pLysS質粒含有表達 T7 溶菌酶的基因，能夠降低目的基因的背景表達水平，但不干擾IPTG誘導的表達，適合表達毒性蛋白和非毒性蛋白。Rosetta-gami(DE3)pLysS 菌株具有卡那霉素，氯霉素，鏈霉素，四環素抗性，經pUC19質粒檢測轉化效率高達108cfu/μg。操 作 說 明1.取100μl冰上融化的Rosetta-gami(DE3)pLysS 感受態細胞，加入目的質粒并輕輕混勻，冰上靜置25分鐘。2.42回冰上并靜置2分鐘，晃動會降低轉化效率。3.向離心管中加入700μl不含抗生素的無菌培養基（2YT或LB），混勻后37℃，200rpm復蘇60分鐘。4.5000rpm離心一分鐘收菌，留取100μl左右上清輕輕吹打重懸菌塊并涂布到含相應抗生素的2YT 或LB培養基上。5.將平板倒置放于37℃培養箱過夜培養。注 意 事 項1. 感受態細胞最好在冰上融化。2.混入質粒時應輕柔操作。3.轉化高濃度的質粒可相應減少最終用于涂板的菌量。4.誘導時，IPTG濃度可選（0.1-2mM均可）。5.為獲得需要量的蛋白，最佳誘導時間，溫度，IPTG濃度需實驗者優化。6.菌株攜帶 pLysS質粒，除復蘇培養基為無抗生素外，其余所用培養基、培養液均應含有34 µg/ml氯霉素，以防質粒丟失。7.具有卡那霉素抗性，不能用于具有卡那霉素抗性質粒的表達。Sample Induction Protocol(for reference only)1.   Inoculate a single colony from a freshly streaked plate into 3ml of LB medium containing the appropriate antibiotic for the plasmid and host strain.2.   Incubate with shaking at 200 rpm at 37℃ overnight. 3.   Inoculate 50 ml of LB medium containing the appropriate antibiotic with 0.5 ml of the overnight culture prepared in step 2(use the 500 ml triangular flask as the container would be better).4.   Incubate with shaking at 150 rpm at 37℃ until the OD 600 reaches 0.5-0.8. (0.6 recommended; about 2.5h).5.   (Optional)Pipet 1ml of  the cultures into clean microcentrifuge tubes and  place the tubes on ice until needed  for gel analysis or storage at  -20℃. These will serve as the non-induced control samples.     6.   Add IPTG to a final concentration of 1 mM.  Optimal time for induction of the target protein may vary from 2-16 hours, depending on the protein.7.   Incubate with shaking at 120 rpm at 37℃ for 2-4 hours. To determine the optimal time for induction of the target protein, it is recommended that a time course experiment be performed varying the induction from 2-16 hours.8.   Place the culture on ice for 10 minutes. Harvest cells by centrifugation at 5,000 x g for 10minutes at 4℃.9.   Remove the supernatant and store the cell pellet at -20℃ (storage at lower temperatures is also acceptable). 

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