发信人: IVYtony (Tony), 信区: Biology
标 题: Re: Question regarding inducible operon
发信站: Unknown Space - 未名空间 (Wed Dec 1 18:56:17 2004) WWW-POST

hmmm, to answer your question, I'd like to start as if you understood the
transcription machinery in prokaryotes (E.coli falls into the category of
prokaryotes). In the lac operon (repressor) experiment, we use the plasmid
construct that has incorporated the selective marker gene (e.g. uracil
gene,etc). The marker gene can tell you whether the transcription occurs or
not, because the media that we use contains no uracil, the E.coli iteself has
to produce the uracil. The plasmid construct is already a loop
double-stranded DNA, so
there's no problem of DNA loop that you mentioned.

In vitro studies, you can purify the proteins and the plasmids as much as you
possibly can. Also, the addition of protein(s) (RNA polymerase, etc) to the in
vitro transcription (txn) reaction is up to you. So I don't think there 's
interference of some other unrelated proteins to your txn experiment.

Pls also understand that I might be wrong answering your question, because I
don't know your research context. Hope that helps. Anyways, pls point out
where I was wrong, if any.

Thanks

【 在 space (排骨教主) 的大作中提到: 】
: 请教一下:
:
: Transcription发生的充分必要条件是什么? 象你们讨论的这个实验, 能作in vitro的
吗?
:
: 我问这个问题的原因是对Lac repressor loop感兴趣. 我读过一个paper, 作者能够改
变
: 这个loop的大小, 然后测量repression level随loop size的依赖关系. 他们发现
: repression level随size减小而增加. 但是传统的看法是,DNA很短的时候应该很硬,
: 很难弯曲形成loop. 所以这个实验是和这个观点抵触的. 但是这个我们最近发表的
: 一个模型吻合.
:
: 问题是他们的实验是in vivo的, 其中有很多不定因素. 比如可能有些protein帮助DNA
: 弯曲. 因此我不能说他们这个实验确定无疑支持我们的模型. 于是想in vitro的重复
: 一下他们的实验. 请问一下难度会有多大(我是学物理的)?
:
: THX
:
: 【 在 IVYtony (Tony) 的大作中提到: 】
: : iac operon or lac operon? I guess it should be lac operon.
: : as of the experiment, you can incubate the E.coli in lactose medium wher
: : e the lactose is the only carbon source for E.coli. In order to survive,
: : the activation of beta-galactosidase transcription occurs. The product of
beta galactosidase can help consume lactose.
: : To make sure or test the txn level of beta-galactosidase, you should do
the
: : beta-galactosidase assy, using ONPG which can react with
beta-galactosidase,
: : producing a kind of yellow products. After that, you test the OP value.
voila!
: : Hope that helps a little bit
: : 【 在 julio (july) 的大作中提到: 】
: : : Hi,
: : : My major is chemistry and I'm taking molecular biology this year by
interest.
: : : Here is the question regarding the iac operon:
: : : If you knew only that beta-galactosidase was induced in the presence of
: : : lactose, but you didn't know whether the mechanism involved initiation
of
: : : transcription or activation of existing enzyme molecules, what
experiment
: : : could you perform to determine the exact mechanism of induction?
: : : Hope I can get to know the general approach to this type of questions
with
: : : your suggestion.
: : : Thanks!
: : : julio
:
:








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※ 修改:·IVYtony 於 Dec 1 18:56:17 修改本文·[FROM: 129.82.]
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