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Mix & Go! XJb (DE3) Autolysis Competent Cells
Mix & Go! XJb (DE3) Autolysis Competent Cells
- 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.
While there are many cell lysis methods available to scientists, unfortunately none of these methods combine all the ideal features for simple, efficient, economical, and gentle lysis of E. coli cells. The E. coli XJ autolysing strains from Zymo Research were engineered to address this problem. Mild expression of a chromosomally encoded bacteriophage λ R gene, encoding the λ lysozyme, also known as λ endolysin, is induced during growth. Cells are harvested intact while the peptidoglycan layer of the cell walls has been protected from digestion by the cytoplasmic membrane. The membrane is, however, amenable to disruption by a brief physico-chemical stress such as a freeze-thaw cycle after harvesting the cells. The XJ Autolysis™ method is highly efficient and takes only minutes (unlike traditional multiple freeze-thaw cycles). It can be applied to any number of samples without increasing processing time and labor (unlike sonication or French-press), is reliable and repeatable (unlike lysozyme treatment), and finally, is fully compatible with a wide range of buffers. Additionally, it does not require use of any potentially interfering components such as detergents, commonly found in various lytic buffers. They are also applicable for nucleic acid purification, and available with a DE3 lysogen encoding the T7 polymerase for expressing recombinant proteins driven by the T7 promoter.
|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?
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.