BL21中质粒不稳定是因为：BL21不像DH5a，JM109那些克隆用菌一样是DNA酶缺陷型，所以时间一长，BL21中的外源质粒有可能会被降解，而克隆用菌中的质粒则相当稳定。

       至于为什么BL21中的外源质粒丢失后，还能在相应抗性的平板上长出来的问题是因为：BL21不是RecA缺陷型。质粒上的抗性基因有可能被整合到菌的基因组上，所以质粒丢失后还能在相应的平板上长出来。

       rosetta菌种本身含有一种质粒，此质粒编码稀有密码子的tRNA，因此如果目的基因中含的稀有密码子较多，用此菌种表达比较合适。

        rosetta菌中的质粒还带有抗氯霉素标记因此rosetta菌本身可以抗氯霉素，但这个一般是用于筛选菌种防止污染，而与原核表达无关。因此表达过程中无需全程都加氯霉素

        RosettaTM宿主菌是BL21衍生菌，能明显增强带有大肠杆菌稀有密码子的真核蛋白的表达。该菌株通过一个相容性氯霉素抗性质粒补充密码子AUA、AGG、AGA、CUA、CCC和GGA的tRNAs。这样Rosetta菌株实现了“万能”的翻译。而一般情况下，由于受大肠杆菌密码子使用频率的限制，真核蛋白表达常有困难。tRNA基因由它们的天然启动子驱动。在Rosetta（DE3）pLysS和Rosetta（DE3）pLacI中，稀有tRNA基因存在于分别带有T7溶菌酶和lac阻遏基因的同一质粒上。

        大量摇菌表达前，先要划板挑若干单菌落做小量诱导表达，在上样量相同的情况下，我们能看到他们的表达量是不同的，保存表达量高的单菌落，尽快大摇表达，如果放时间过久，还得划板挑克隆选取表达高的单菌落大摇（虽然都来源于一个单克隆菌，但是划板后每个克隆表达量又有不同，原因不明，望大家赐教），这样能尽可能得到高表达。

        另外，选Rosetta的原因，是因为它能满足一些稀有密码子的表达，BL21则不行（导致有些蛋白不表达），所以我们有时候不得不选前者。毕竟能出结果是最重要的

至于Rosetta为何不适宜用于工业化生产，其实与它的表达量有关的，试想一个菌株中拥有N多的质粒或表达框架在里面，那么同样的表达质粒导入，而在别的菌株中就只有一个表达质粒在里面，在相同的培养与诱导条件(即提供的材料量一样)下，可以想像Rosetta菌株中期望的表达质粒能用到的材料量占多少？那么你期望的表达量就肯定不会够了。至于我为何这么说，我已同时做了BL21(DE3)和Rosetta(DE3)为宿主进行表达我的目的基因，就会发现BL21(DE3)为宿主时表达量就会很大，相对来说Rosetta就小了。

the Rossetta strain are designed to enchance the expression of eukaryotic protein that contain codons rarely used in E.Coli. Expression of such protein can be dramatically increased when the level of rare tRNA is increased within the host. Rossetta strains supply tRNA for the codons AUA,AGG,AGA,CUA,CCC and GGA on a plasmid. if your target gene is eukaryotic protein and contain that kinda six rare codons, it will be very useful for you!

Rosetta-gami? Host StrainsRosetta-gami host strains are derivatives of Origami (a K-12 strain), and are designed for enhanced cytoplasmic disulfide bond formation plus enhanced expression of eukaryotic proteins that contain codons rarely used in E. coli. These strains carry all of the characteristics of the Origami strain, plus they supply tRNA genes for AGG, AGA, AUA, CUA, CCC, GGA on a ColE1-compatible chloramphenicol-resistant plasmid. Thus the Rosetta-gami strains provide for "universal" translation which is otherwise limited by the codon usage of E. coli. Transcription of the tRNA genes is driven by their native promoters. In Rosetta-gami(DE3)pLysS and Rosetta-gami(DE3)pLacI, the rare tRNA genes are present on the same plasmids that carry the T7 lysozyme and lac repressor genes, respectively. The designation (DE3) indicates that the host is a lysogen of ?DE3, and therefore carries a chromosomal copy of the T7 RNA polymerase gene under control of the lacUV5 promoter, making this strain suitable for protein expression using vectors containing the T7 or T7lac promoter. In Rosetta-gami 2(DE3)pLysS, the rare tRNA genes are present on the same plasmid that carries the T7 lysozyme gene. T7 lysozyme is a natural inhibitor of T7 RNA polymerase and serves to suppress the activity of T7 RNA polymerase prior to induction, thus stabilizing recombinants encoding target proteins that affect cell growth and viability. Hosts containing the trxB and gor mutations are only recommended for the expression of proteins that require disulfide bond formation for proper folding. Proteins which do not require disulfide bonds may have reduced solubility in these strains due to intermolecular disulfide bonds.

Origami B Origami B host strains carry the same trxB/gor mutations as the original Origami strains, except that they are derived from a lacZY mutant of BL21. Thus the Origami B strains combine the desirable characteristics of BL21, Tuner? and Origami hosts in one strain background. The trxB and gor mutations are selectable on kanamycin and tetracycline, respectively; therefore, these strains are recommended for use only with pET plasmids carrying the ampicillin resistance marker bla.

Origami? 2 host strains are K-12 derivatives that have mutations in both the thioredoxin reductase (trx and glutathione reductase (gor) genes, which greatly enhance disulfide bond formation in the cytoplasm. Unlike the original Origami strains, the Origami 2 strains are kanamycin sensitive, making these host strains compatible with any Novagen expression vector. The gor mutation is still selected for by tetracycline as in the original strains.

RosettaBlue? RosettaBlue host strains are NovaBlue derivatives that combine high transformation efficiency and recA endA lacIq mutations with enhanced expression of eukaryotic proteins that contain codons rarely used in E. coli. These strains supply tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons on a compatible chloramphenicol-resistant plasmid. The tRNA genes are driven by their native promoters. In RosettaBlue(DE3)pLysS and RosettaBlue(DE3)pLacI, the rare tRNA genes are present on the same plasmids that carry the T7 lysozyme and lac repressor genes, respectively. Blue/white screening is not possible with RosettaBlue(DE3) strains due to the presence of lacZ a-peptide coding sequences in the DE3 lysogenic phage.

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