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 (Wed Sep 18 13:46:28 2002) WWW-POST

IPTG induces the expression of T7 polymerase (T7 pol is under the control of
lac, inducible by IPTG), which in turn translates your gene of interest
because it is under the control of T7 promoter and has to be processed by T7
pol, but not E.coli pol. without IPTG, T7 pol has very low basal expression
level, and your gene of interest will have some low basal expression too.
plysS expresses a low level of lysozyme, which functions as an inhibitor for
T7 pol, and thus, with no IPTG induction, inhibits basal expression of your
gene, preventing cell death due to the toxicity of your gene. when IPTG is
added, due to large quantity of T7 pol being expressed, the low level of
lysozyme is overwhelmed and can be ignored, leading to high expression of your
gene.
I am not sure if you kind of misunderstood the system. Hope this is helpful.
and IP = immunoprecipitation: to use Ab to affinity pull down your targeted
protein. you can use Abs anti-the tag on your protein, or you can use Abs to
target the protein itself.


【 在 aoexy (lili) 的大作中提到: 】
: 【 在 leohawk (leohawk) 的大作中提到: 】
: : I had very bad experence with this line, wasted one year of my work in one
: : project... problem is it produce another protein under IPTG induction,
which
:
: : of the same size of my protein and weirdly, co-purify with it.
: :
: : co-purified protein is the lysozyme, it helps to break the cell. I forgot
: : how it reduce background expression... think hard...probably by blocking
: : trivial T7 polymerase creation without IPTG, which result in no
: transcription
: : of your protein.
: ~~~~~~~~~~ ~~~~~~~~~~~
: this is my problem now. Because my protein is not expressed in PET
: vector, and it is under the constitutive control of T7 promotor instead of
: being induced by IPTG. so, I am afraid that my protein will be blocked or
: degraded as back ground expression. But I checked the promega comepetent
cells
: catolog and can't find another host strain for translation of my vector.
:
: and thanks leohawk for sharing your experience with me.
:
: : BL21DE3 is good for expression of protein in PET vector, I think it is a
: : pretty good system.
: :
: : 【 在 aoexy (lili) 的大作中提到: 】
: : : Promega 得BL21 cell， discription 这样说：Bl21(DE3)pLysS can be used
with
: : : protein expression vectors that are under the control of the T7
promoter,
: such
: : : as pET vectorers. This strain is lysogenic for lambda-DE3(4), which
: contains
: : : the T7 bacteriophage gene1, encoding T7 RNA polymerase under the control
: of
: : : the lac UV5 promoter. BL21(DE3) pLysS also contains the pLysS palsmid,
: which
: : : carries the gene encoding T7 lysozyme. T7 lysozyme lowers the background
: : : expression level of target genes under the control of the T7 promoter
but
: does
: : : not inerfere with the level of expression achieved following induction
: with
: : : IPTG.
: : : 如果我没有理解错的话，依照这一段话 的意思，如果一个Vector 的fusion
protein
: : : 是constitutive 表达在T7 promoter 下 的话，是不是就会被降解掉？如果是这样
，
: 各位
: : : 大侠可否给我推荐一个合适的host stain for constitutive expression of
target
: : : gene under the control of T7 promoter without using the induction of
IPTG?
: : : 另外，再请教一下，所谓的IP是不是就一有两个tag的western?
: : : 谢谢大家先 。
: :
: :
:
:

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