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Sauer:Lysing E. coli with Lysozymes: Difference between revisions
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Sauer:Lysing E. coli with Lysozymes (view source)
Revision as of 08:08, 29 February 2008
, 29 February 2008→Intracellular Lysozymes
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One problem with egg lysozyme is that it doesn't seem to work well at high Mg++ concentrations, in certain buffers, or in cells that are in stationary phase. So, if you want to make a lysate in a defined buffer (like ribosomes in 10 mM Mg++), lysis is greatly impeded. To fix this, I made a new "lysis plasmid" inspired by the pLysS/E plasmids sold by Novagen. pLysS constitutively expresses the T4 lysozyme. It is intended to inhibit the activity of T4 polymerase in T4 expression systems. It has the added benefit that disruption of the inner membrane allows the lysozyme to get to the cell wall and lyse the cells. So, freezing and thawing without a cryoprotectant or adding chloroform to the cells causes lysis. The problem with pLysS is that cells harboring it are sick and lyse during centrifugation, the lysozyme is not a dedicated lysozyme (it has other cellular functions), and that it doesn't seem to lyse stationary phase cells well. So, after speaking with Ryland Young, I decided to make a better plasmid. | One problem with egg lysozyme is that it doesn't seem to work well at high Mg++ concentrations, in certain buffers, or in cells that are in stationary phase. So, if you want to make a lysate in a defined buffer (like ribosomes in 10 mM Mg++), lysis is greatly impeded. To fix this, I made a new "lysis plasmid" inspired by the pLysS/E plasmids sold by Novagen. pLysS constitutively expresses the T4 lysozyme. It is intended to inhibit the activity of T4 polymerase in T4 expression systems. It has the added benefit that disruption of the inner membrane allows the lysozyme to get to the cell wall and lyse the cells. So, freezing and thawing without a cryoprotectant or adding chloroform to the cells causes lysis. The problem with pLysS is that cells harboring it are sick and lyse during centrifugation, the lysozyme is not a dedicated lysozyme (it has other cellular functions), and that it doesn't seem to lyse stationary phase cells well. So, after speaking with Ryland Young, I decided to make a better plasmid. | ||
In my construct, I placed the phage Lambda lysozyme "R gene" under constitutive control of the moderate Bla promoter from beta-lactamase. This is a promoter that appears to exhibit even expression throughout growth (reference coming, some RNA micro-array paper). Cells harboring this plasmid grow well and can be frozen if there is at least 10% glycerol present. To lyse these cells, I add 10 uL of chloroform per mL of liquid and vortex. At room temp, the cells completely lyse in about a minute. | In my construct, I placed the phage Lambda lysozyme "R gene" under constitutive control of the moderate Bla promoter from beta-lactamase. This is a promoter that appears to exhibit even expression throughout growth (reference coming, some RNA micro-array paper). Cells harboring this plasmid grow well and can be frozen if there is at least 10% glycerol present. To lyse these cells, I add 10 uL of chloroform per mL of liquid and vortex. At room temp, the cells completely lyse in about a minute. Cells harboring the lysis plasmid can be transformed with chemical methods (CsCl, TSS, etc.) but NOT by electroporation. | ||
[[Category:Protocol]] | [[Category:Protocol]] | ||
