Highlights
- Fast: 80 - 90% of E.coli are lysed in only 10 minutes after harvesting.
- High Transformation Efficiencies: Achieve 108 - 109 transformants per µg of plasmid DNA.
- DE3 Lysogen: Encodes the T7 polymerase for expressing recombinant proteins under the control of the T7 promoter.
Description
| Autolysis | XJb lysis efficiency is 10-20 % lower than XJa. For optimal lysis, more care needs to be taken when selecting the lysis buffer. However, even very low concentrations of detergent may improve lysis significantly. |
|---|---|
| Cell Growth | A very robust strain, reaching higher OD’s than E. coli K-strains. |
| DNA Extraction | XJb is not optimal for DNA extraction. |
| DNA Stability | This strain is RecA positive. |
| Genotype | F- ompT hsdSB(rB - mB -) gal dcm ΔaraB::λR, cat (CmR), λ(DE3) |
| Processing Time | 10 minutes |
| Product Storage | -70°C to -80°C |
| Protein Expression | XJb is ideal for recombinant protein expression. It lacks Lon and OmpT proteases, leading to higher protein yields. |
| Transformation Efficiency | 108 - 109 transformants per µg of plasmid DNA |
Q1: Can glucose be added to the growth media?
When glucose is added to the growth media, it inhibits the induction of the autolysis genes when it is present in the media. As the cells grow, they consume the glucose as a carbon source. Once the glucose has been consumed autolysis begins.
Q2: Can glycerol be present during the freeze-thaw cycle?
Do not perform the freeze and thaw cycle in a buffer containing glycerol. Glycerol protects the E.coli from forming ice crystals which are essential to the lysis of the cells.
Q3: What if the lysate is extremely viscous?
Depending on the amount of material used, the lysed material may become viscous, preventing efficient manipulation. However, for most applications it is not necessary to use a large amount of cell material. If necessary, vortexing vigorously for 30 seconds will decrease viscosity in most cases. Alternatively, a nuclease treatment (e.g. DNAse I) can be used to reduce viscosity. Diluting the cell lysate with additional buffer will also reduce viscosity issues.
Q4: How do you improve lysis efficiency?
If the results obtained are not satisfactory, lysis can be significantly improved by incubating the cells at higher temperatures (25 - 37°C) or for longer time (10 or 20 minutes) after thawing (step 5).
Q5: Do heat shock and outgrowth steps have to be performed when transforming XJb Autolysis E. coli?
It is necessary for high transformation efficiencies.However, if your experiment does not require very high transformation efficiency (e.g. when using plasmid stock to transform E. coli), incubate the DNA and cells on ice for 1-5 minutes and spread directly onto prewarmed plates.
Q6: Is a starter culture necessary?
For best results, cells should not be growing actively prior to arabinose induction. This is achieved by using an overnight starter, where cells are already in the stationary growth phase, as stated in the protocol. If a fresher starter needs to be used, include arabinose already in the starter culture.
Q7: Will chitin be degraded?
Non-λ lysozyme usually is able to degrade chitin. However, the λ lysozyme expressed in these cells is not able to degrade chitin. λ lysozyme is a transglycosylase.
Q8: What buffer should the cell pellet be resuspended in?
Resuspend the cell pellet in water with or without 0.01% - 0.1% Triton X-100. For His-tag purification, resuspend in the His-Binding Buffer of the His-spin Protein Miniprep kit (Zymo Research product # P2001 or P2002). Acidic buffers and buffers containing higher concentrations of Mg2+ (>1 mM), and related metals that stabilize cell walls, inhibit lysis reaction to a various extent. If possible, add magnesium to the buffer after cells are lysed.
Q9: Are competent cells GMOs?
All our competent cells are classified into Biosafety level 1 and are not genetic modified organisms. Only when transformed with a plasmid they become GMOs.
Q10: Are the Mix & Go strains dam+ and dcm+?
Most cloning strains will be dam+/dcm+ unless specifically noted in the genotype.
Q11: Do the Mix & Go strains methylate DNA?
Yes
Q12: Which strains are equivalent to the Zymo strains?
DH5α is equivalent to Zymo 5α. DH10B, Top10, and One Shot Top10 are equivalent to Zymo 10B.For XL-21 Blue, JM109 is the closest match and for Stbl3, HB101 is the closest match.
Q13: How to reduce satellite colonies on agar plates?
– Prepare fresh agar plates– Use more antibiotics in plates– Incubate plates for a shorter time after plating cells
Q14: Is it possible to dilute the competent cells?
We do not recommend diluting the competent cells. We recommend using less DNA to transform cells, or aliquot cells in smaller volumes before transformation. If absolutely necessary, cold 1X Competent Buffer (Mix & Go Transformation Kit, T3001 & T3002) should be used in the dilution.
Q15: Which antibiotics can be used with the Mix & Go! procedure?
No outgrowth is necessary when using Ampicillin or Carbenicillin for selection. However, an outgrowth step is required when using Chloramphenicol, Kanamycin, and Tetracycline because of the mode of action of the antibiotic itself. We recommend the following procedure for the outgrowth step:1. Incubate cells on ice for 5-10 min after addition of plasmid.2. Add 4 volumes of SOC media.3. Incubate at 37°C for 60 min with gentle shaking at 200-300 rpm.4. Spread on a pre-warmed culture plate containing the appropriate antibiotic.
Q16: Which Plasmid Size can be used for transformation?
For Zymo 5α and Zymo 10B up to 20kb. However, transformation efficiency decreases proportionally from 10-20kb. Above 20kb, cells are difficult to transform. JM109, HB101, XJa, XJa (DE3), XJb, XJb (DE3) and TG1 can handle constructs up to 10kb.
Q17: Which is the recommended DNA concentration and volume for transformation?
There really is no maximum or minimum recommended DNA concentration, but we use 10 pg for quality control. However, the volume of DNA added should not exceed 5% of the cells total volume; the efficiency can decrease several fold as the volume of DNA used increases. If the DNA sample is too diluted, use our DNA Clean & Concentrator.
Q18: What are some tips to improve transformation efficiency?
1. Thaw cells on ice, not room temperature.2. Incubate cells and DNA mixture on ice, not at room temperature. However, do not incubate longer then 1 hour.3. Ensure cells are still frozen when received.4. Pre-warm the culture plates at 37°C for at least 30 minutes.5. Prepare fresh LB agar plates containing the appropriate antibiotic. 6. Prepare a new DNA sample.7. Store the cells at -80°C (not 4°C or -20°C). If the freezer breaks, the cells should be OK as long as the temp does not go higher than -50°C.8. Avoid freeze/thaw cycles.
Q19: How will a heat-shock affect my Transformation Efficiency?
Heat shock is not necessary, however sometimes it can be beneficiary when preparing libraries or transforming XJb Autolysis E. coli strains.We recommend the following protocol for Heat Shock with Outgrowth: 1. Incubate cells on ice for 5-10 min after addition of plasmid. 2. Incubate cells at 42°C for 45 seconds.3. Add 450 ml of SOC to the cells. 4. Incubate at 37°C for 60 min with gentle shaking at 200-300 rpm.5. Spread on a pre-warmed culture plate containing the appropriate antibiotic.
To clone new GFP-like fluorescent proteins from Obelia medusa, the authors identified the potential genes using expression libraries and cloned the genes into a vector. Expression of the proteins was facilitated by using XJb Autolysis E. coli cells from Zymo Research. The authors were able to purify three proteins from Obelia medusa that fluoresce in three different colors: cyan, green, and yellow.
Aglyamova, G.V. et al. (2011) Multi-colored homologs of the green fluorescent protein from hydromedusa Obelia sp. Photochem Photobiol Sci (8):1303-9.ebiomall.com
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抗原与其所诱导产生的抗体或致敏淋巴细胞特异性结合的能力。抗原性的强弱与抗原分子的大小、化学成分、抗原决定簇的结构、抗原与被免疫动物亲缘关系的远近等有密切关系。通常认为抗原的分子量愈大、化学组成愈复杂、立体结构愈完整以及与被免疫动物的亲缘关系愈远，则抗原性愈强。抗原的物理状态也对抗原性发生影响，例如蛋白质，聚合状态的比单体的抗原性强，一般球形分子的比纤维形分子的抗原性强。抗原加入佐剂改变物理状态后，抗原性也得到增强。例如，分子量高达10万的明胶由于缺乏苯环氨基酸，稳定性较差，在进入机体后容易被酶降解成低分子物质，如果加入少量酪氨酸（苯环氨基酸），就能增强其抗原性。
中文名：抗原性
外文名：antigenicity
别称：免疫反应性
抗原性介绍：流感病毒按抗原性
抗体是属于у-球蛋白，或是淋巴细胞，都是对某种抗原反应而形成的；它附着在抗原上，起着抑制抗原的作用。如果抗体完全抑制了抗原，即使我们感染了病毒或细菌，也会在不知不觉中战胜疾病。相反，当抗原势力超过抗体时，疾病则会恶化。
一旦体内产生抗体时，当下次相同的病原体侵入时，会比前次更快发动免疫机构，将疾病防患于未然。许多病毒性疾病患者，经过一次治疗后便不再发病，是因为身体内已有了免疫的机构。
抗体指机体的免疫系统在抗原刺激下，由B淋巴细胞或记忆细胞增殖分化成的浆细胞所产生的、可与相应抗原发生特异性结合的免疫球蛋白。
这血清里含有抗体。当然也含有抗原，可以刺激机体产生免疫反应
抗原在体内可以引起免疫反应。抗体是由抗原刺激机体产生的，能与抗原特异性结合是抗原失去毒性的球蛋白。
也就是说，该教授感染的sars病毒是抗原。使他患病，而另一个已痊愈的非典病人，由于感染过非典，体内产生过抗体。这时候抗体就在病人的血清中，该教授用权与病人的血清注射相当于注射了抗体，于是抗体消灭了抗原，并就好了。
免疫的过程比较复杂，一般是这样的。
抗原经过吞噬细胞处理之后，露出了抗原决定簇，抗原决定簇决定了抗原的特异性。
然后吞噬细胞将抗原信息船体给T淋巴细胞，然后T淋巴细胞吧抗原信息传递给B淋巴细胞。这时候B淋巴细胞接受刺激，分裂分化成效应B细胞，和记忆细胞。效应B细胞立刻合成有关抗体，使抗原发生特异性凝结，就是抗原失效，然后再有吞噬细胞等吧抗原消灭。
另外当相同抗原再次刺激机体是，记忆细胞就会直接分化成效应b细胞，分泌抗体，消灭抗原，这个过程叫做二次免疫。我们的疫苗应用的就是二次免疫效果迅速强烈的原理。
当然也有一些抗原不需要T细胞，直接有吞噬细胞处理后呈递给B细胞。
看了百科以后依然不太明白，语言通俗一点最好。谢谢。
我们自身的细胞，蛋白也是抗原，但是自体在正常情况下没有针对自身的抗体。抗体产生的过程很复杂，我简单说一下：抗体由B细胞产生。本质是蛋白质。B细胞在成熟的过程中，编码抗体抗原识别区的那段基因会发生随机组合，这样就有可能会产生无数种抗体。但是如果一个B细胞产生了针对自身的抗体，那这个细胞就会和抚养它的细胞发生过于紧密的接触，被杀死。如果一个B细胞产生的抗体还不能识别任何抗原，那它就得不到足够的生长刺激，所以也不能存活。最后成熟的免疫细胞只占很小一部分。只有那些可以识别外源抗体的B细胞才能得到刺激，大量增殖。然后体内的抗体就增高了。
所以你的最后一句话不对。并不是人人都可以对任何抗原产生抗体的。比如青霉素过敏，过敏的人就是因为体内有针对青霉素的抗体IgE。而其他人就没有抗体。
B细胞的抗原受体就是镶嵌在脂质双层中的膜结构蛋白，叫做膜表面免疫球蛋白（SmIg）。每个淋巴细胞克隆，只具有识别某一抗原决定簇的受体，每个淋巴细胞也只能产生某一种特异性抗体，这也是单克隆抗体只针对某一种抗原决定簇反应的基础。体内有千万种淋巴细胞克隆，所以机体能对各种不同的抗原性异物进行识别，从而发生免疫应答。
T细胞的抗原受体（T cell receptor, TCR）与CD3呈复合物形式存在于抗原特异性T细胞表面。在识别靶细胞过程中还有赖于非特异性的其他表面分子辅助，这些分子主要包括CD4、CD8、MHCI、MHCII类分子、淋巴细胞功能相关抗原-1（LFA-1）、细胞间黏附分子-1（ICAM-1，即CD54）、LFA-2（CD2）和 LFA-3（CD58）。TCR的多肽链是异质的，根据抗原结构和编码基因不同，已发现有a, b, g和d四种多肽链，据其组成不同，可分为两种类型，即abTCR和gdTCR。大多数TCR是abTCR，其中CD4+abTCR T细胞可识别非己MHCII类抗原（同种异体抗原）或自身MHCII类抗原与外来抗原复合物。abTCR识别外来抗原必须经过抗原提呈细胞（antigen presenting cell, APC）的加工处理，在外来抗原与APC表面MHC抗原结合成复合物才能被Th细胞识别。少数TCR由gd链组成，对gdTCR在体内的生理功能尚未完全清楚。
