Sauer:Lysing E. coli with Lysozymes: Difference between revisions
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===Hen Egg White Lysozyme=== | ===Hen Egg White Lysozyme=== | ||
I prepare the lysozyme fresh each time from lyophilized powder. Pre-warm the bottle to prevent moisture from condensing in the bottle when it's opened. Pure B-Per doesn't seem to allow it into solution and it remains mostly as an inactive precipitate. So I make a ~10 mg/mL stock in some buffer off my shelf (I usually use 50 mM bis-Tris, pH 6.5). Then I dilute from this to 0.1 to 0.01 mg/mL in the B-Per lysis solution. To save time, I use a pipette tip and lift out dried lysozyme horizontally from the bottle, I then place a microfuge tube over the tip and shake off the powder. I measured the before and after weight of several tubes and found that I can make pretty close to a 10 mg/mL solution by just adding 400 | I prepare the lysozyme fresh each time from lyophilized powder. Pre-warm the bottle to prevent moisture from condensing in the bottle when it's opened. Pure B-Per doesn't seem to allow it into solution and it remains mostly as an inactive precipitate. So I make a ~10 mg/mL stock in some buffer off my shelf (I usually use 50 mM bis-Tris, pH 6.5). Then I dilute from this to 0.1 to 0.01 mg/mL in the B-Per lysis solution. To save time, I use a pipette tip and lift out dried lysozyme horizontally from the bottle, I then place a microfuge tube over the tip and shake off the powder. I measured the before and after weight of several tubes and found that I can make pretty close to a 10 mg/mL solution by just adding 400 μL of buffer to the tube. So, I no longer weigh out lysozyme each time, I just assume that my tip carries about 4 mg of protein. | ||
In most cases, simply adding lysozyme to your resuspended cells and waiting is sufficient to invoke lysis. Some protocols call for placing the cells on ice during lysis in an effort to curb proteolytic activity. This never made much sense to me, you also slow down the activity of the lysozyme. I have never seen a comparison of the activities of proteases and lysozyme at 0 and 25 degrees, but I generally lyse at room temperature. | |||
Lysis is apparent by a reduction in turbidity and a severe increase in viscosity from the liberated chromosomes. For protein preparations, I usually add Benzonase because it has activity toward double- and single-stranded DNA and RNA. If you just add DNase, be aware that most are purified from pancreas and are loaded with proteases. If you see a lot of damage to your protein during lysis, add proteiease inhibitiors and/or change nuclease. | |||
===Intracellular Lysozymes=== | ===Intracellular Lysozymes=== | ||
Revision as of 14:40, 24 January 2007
Submitted by Sean Moore
Getting The Most Out Of Your Bugs
Hen Egg White Lysozyme
I prepare the lysozyme fresh each time from lyophilized powder. Pre-warm the bottle to prevent moisture from condensing in the bottle when it's opened. Pure B-Per doesn't seem to allow it into solution and it remains mostly as an inactive precipitate. So I make a ~10 mg/mL stock in some buffer off my shelf (I usually use 50 mM bis-Tris, pH 6.5). Then I dilute from this to 0.1 to 0.01 mg/mL in the B-Per lysis solution. To save time, I use a pipette tip and lift out dried lysozyme horizontally from the bottle, I then place a microfuge tube over the tip and shake off the powder. I measured the before and after weight of several tubes and found that I can make pretty close to a 10 mg/mL solution by just adding 400 μL of buffer to the tube. So, I no longer weigh out lysozyme each time, I just assume that my tip carries about 4 mg of protein.
In most cases, simply adding lysozyme to your resuspended cells and waiting is sufficient to invoke lysis. Some protocols call for placing the cells on ice during lysis in an effort to curb proteolytic activity. This never made much sense to me, you also slow down the activity of the lysozyme. I have never seen a comparison of the activities of proteases and lysozyme at 0 and 25 degrees, but I generally lyse at room temperature.
Lysis is apparent by a reduction in turbidity and a severe increase in viscosity from the liberated chromosomes. For protein preparations, I usually add Benzonase because it has activity toward double- and single-stranded DNA and RNA. If you just add DNase, be aware that most are purified from pancreas and are loaded with proteases. If you see a lot of damage to your protein during lysis, add proteiease inhibitiors and/or change nuclease.
Intracellular Lysozymes
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.
