发信人: forrestgump (sleepless in Houston), 信区: Biology
标 题: Re: 请教E.coli competent cells
发信站: The unknown SPACE (Thu Sep 19 18:54:30 2002) WWW-POST

I do not think you fully understood my answer to your question yet. let me put
it this way.
what kind of BL21 are you using? BL21 has BL21 (DE3), (DE3) pLysS, ...
different subtypes. I assume that you are using DE3 related strains. DE3
strain has a lambda phage lysogenized in BL21 e.coli, on which there is a T7
polymerase gene under the control of lac promoter. when you grow the cells up,
no or very little T7 pol can be expressed. when you add IPTG, it induces the
expression of T7 pol, which then acts on the T7 promoter on your gene of
interest and induces its expression.
so, in other words, if you are not using DE3 or if you do not add IPTG, there
will never be any expression of your protein (or very very little, due to the
leakage of T7 pol expression). E.coli does not have endogenous T7 pol, and
therefore your gene cannot be constitutively expressed unless IPTG is added.
it is very unlikely that the lysozyme is causing your problem.
now, let's see what is causing the lack of expression.
the most common reason is frameshift. are u 100% sure that your gene is in
frame with the tag ATG?
how toxic is your gene? do the cells grow normally before and after induction?
how do you know that your gene is not lost during expression?





【 在 aoexy (lili) 的大作中提到: 】
: 小石头，谢谢你。
: my vecotors are the vectors which are used in yeast two hybrid system.
pGADT7
: with Ha tag and pGBKT7 with myc tag. The target gene is directly under the
: control of T7 promoter which is not suppressed by lacZ, so they are
: constitutively expressed and need not be induced by IPTG. In E.coli, I use
: the BL21 as host strain. Now the problem is that I could not get any band in
: western blot. I am now wondering whether the fusion gene is correctly
: expressed. All vectores are sequenced and should be right. In protocol they
: recommend in vitro translated with S radiolabelled protein.

--
LIfe is like a box of chocolate.
You never know what is going to be the next.

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