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T7 Express lysY Competent E. coli (High Efficiency) > FAQ |  | T7 Express lysY Competent E. coli (High Efficiency) FAQ
See the Competent Cells FAQ also.
Q1: Why are there no colonies or no growth in liquid culture (C3010)?
Q2: Why is there no protein visible on gel or no activity (C3010)?
Q3: Why is induced protein insoluble (C3010)?
Q4: What are the solutions/recipes (C3010)?
Q5: What are the strain properties (C3010)?
Q1: Why are there no colonies or no growth in liquid culture (C3010)?
A1: Even though T7 expression is tightly regulated, there may be a low level of basal expression in the T7 Express host. If toxicity of the expressed protein is likely, transformation of the expression plasmid should be carried out in one of the following strains:
» T7 Express Iq : over-expression of the LacI repressor reduces basal expression of the T7 RNA polymerase
» T7 Express lysY : lysY produces mutant T7 lysozyme which binds to T7 RNA polymerase, reducing basal expression of the target protein. Upon induction, newly made T7 RNA polymerase titrates out the lysozyme and results in expression of the target protein
» T7 Express Iq/lysY combines both above effects.
Incubation at 30°C or room temperature may also alleviate toxicity issues. In addition, check antibiotic concentration (test with control plasmid).
Q2: Why is there no protein visible on gel or no activity (C3010)?
A2: Check for toxicity - no protein may mean the cells have eliminated or deleted elements in the expression plasmid.
Culture cells for protein induction. Just before induction, plate a sample on duplicate plates with and without antibiotic selection. If toxicity is an issue, there will be a significant difference between the number of colonies on the plates. Fewer colonies will be seen on plates containing antibiotic (indicating that the plasmid has been lost) compared to plates without antibiotic.
Q3: Why is induced protein insoluble (C3010)?
A3: Check for insolubility - this is important because T7 expression often leads to very high production of protein that can result in the target protein becoming insoluble. Solutions around this are:
» Induce at lower temperatures (as low as 12 - 15°C overnight)
» Reduce IPTG concentration to 0.01 - 0.1 mM
» Induce for less time (as little as 15 minutes)
» Induce earlier in growth (OD600 = 0.3 or 0.4)
Q4: What are the solutions/recipes (C3010)?
A4: SOB:
2% Vegetable peptone (or Tryptone)
0.5% Yeast Extract
10 mM NaCl
2.5 mMKCl
10 mM MgCl2
10 mM MgSO4
SOC:
SOB + 20 mM Glucose
LB agar:
1% Tryptone
0.5% Yeast Extract
0.17M NaCl
1.5% Agar
Q5: What are the strain properties (C3010)?
A5: The properties of this strain that contribute to its usefulness as a protein expression strain are described below. The genotypes underlying these properties appear in parentheses.
T7 RNA Polymerase (lacZ::T7 gene1) T7-Express has the T7 RNA polymerase gene inserted into the lac operon on the E. coli chromosome and is expressed under the control of the lac promoter. This configuration provides controlled induction of the polymerase and consequently, inducible control of transcription of genes downstream of the T7 promoter. This system provides potential advantages over strains such as BL21(DE3), that carry the T7 RNA polymerase on a lysogenic prophage. Although λDE3 is normally dormant in the host chromosome, the induction of the SOS cascade can occur as the result of expressing proteins that damage the E. coli chromosome, either directly or indirectly. This may lead to cell lysis.
T7 Lysozyme (lysY): This strain expresses T7 lysozyme variant K128Y which lacks amidase activity, yet retains the ability to inhibit T7 RNA polymerase. Basal expression of the target gene is minimized without inhibiting IPTG-induced expression. The lysY gene is carried on a single-copy miniF plasmid.
Protease Deficient ([lon] ompT): E. coli B strains are "naturally" deficient in the lon protease which in K-12 strains serves to degrade misfolded proteins and to prevent some cell cycle-specific proteins from accumulating. The OmpT protease resides at the surface of wild type E. coli in both K-12 and B strains, presumably helping the cells to derive amino acids from their external environment. Cells deficient in both these proteases are much more amenable to the production of proteins from cloned genes.
Recovery from DNA Damage (sulA11): E. coli cells can tolerate a substantial amount of chronic DNA damage as long as repair is allowed to proceed. This capacity is compromised if the cells are unable to divide following repair. In lon- cells, SulA, a cell division inhibitor, accumulates and causes cells to become hypersensitive to DNA damage. The sulA mutation introduced into the T7 Express strain allows cells to divide more normally in the absence of Lon protease.
Endonuclease I Deficient: (endA) The periplasmic space of wild type E. coli cells contains a nonspecific endonuclease. Extreme care must be taken to avoid degradation of plasmids prepared from these cells. The endA mutation deletes this endonuclease and can significantly improve the quality of plasmid preparations.
Restriction Deficient: (Δ(mcrC-mrr)114::IS10) Wild type E. coli B strains carry a Type I restriction endonuclease which cleaves DNA with the site TGA(N8)TGCT. While E. coli DNA is protected from degradation by a cognate methyl-transferase, foreign DNA will be cut at these sites. The deletion described above eliminates both the methylase and the endonuclease.
Methyl Restriction Deficient: (Δ(mcrC-mrr)114::IS10 and R(mcr-73::miniTn10--TetS)2): E. coli has a system of enzymes encoded by mcrA, mcrBC and mrr which will cleave DNA with methylation patterns found in higher eukaryotes, as well as some plant and bacterial strains. All three Mcr enzymes and Mrr have been inactivated in T7 Express allowing the introduction of eukaryotic DNA of genomic origin (e.g. primary libraries) if desired.
T1 Phage Resistant: (fhuA2) T1, an extremely virulent phage requires the E. coli ferric hydroxamate uptake receptor for infectivity. Deletion of this gene confers resistance to this type of phage, but does not significantly affect the transformation or growth characteristics of the cell.
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