s******y
发帖数: 28562 | 1 我们需要在酵母里同时高表达两个蛋白，但是不知道mamalian常用的那些IRES, P2A
sequence这些东西能不能用在这种场合上？
我们本来想找两个抗性不一样的表达质粒各自表达一个，但是我们能找到的都是URA
marker.这样会弄得很麻烦因为抗性没有差别。 |
I*****y
发帖数: 6402 | 2 maybe you can take a look at pRS plasmids (pRS426 for example), the plasmids
have bi-directional Gal1 promoter that can drive the overexpression of two
genes simultaneously. The selection of markers include any flavors as you
like.
if you use two plasmids, each carrying one gene for expression, you will
have a hard time growing up the cells in double-dropout media. It could work
but at the sacrifice of efficiency.
【在 s******y 的大作中提到】
: 我们需要在酵母里同时高表达两个蛋白，但是不知道mamalian常用的那些IRES, P2A
: sequence这些东西能不能用在这种场合上？
: 我们本来想找两个抗性不一样的表达质粒各自表达一个，但是我们能找到的都是URA
: marker.这样会弄得很麻烦因为抗性没有差别。
|
s******y
发帖数: 28562 | 3 That is correct, we actually don't really like the double selection idea.
If we can have something to drive two genes simultaneously it would be ideal
.
plasmids
two
work
【在 I*****y 的大作中提到】
: maybe you can take a look at pRS plasmids (pRS426 for example), the plasmids
: have bi-directional Gal1 promoter that can drive the overexpression of two
: genes simultaneously. The selection of markers include any flavors as you
: like.
: if you use two plasmids, each carrying one gene for expression, you will
: have a hard time growing up the cells in double-dropout media. It could work
: but at the sacrifice of efficiency.
|
s******y
发帖数: 28562 | 4 不好意思，我去addgene 看了半天，没有找到哪个pRS426是可以同时表达两个蛋白的，
能不能麻烦你再具体一点？
plasmids
two
work
【在 I*****y 的大作中提到】
: maybe you can take a look at pRS plasmids (pRS426 for example), the plasmids
: have bi-directional Gal1 promoter that can drive the overexpression of two
: genes simultaneously. The selection of markers include any flavors as you
: like.
: if you use two plasmids, each carrying one gene for expression, you will
: have a hard time growing up the cells in double-dropout media. It could work
: but at the sacrifice of efficiency.
|
h**********r
发帖数: 671 | 5 必须是pESC系列。
当时我还问大家要不要加kozak序列，也没说出个所以然来。后来我终于等到了客服的
答复。下面就是：
The GAL1 and GAL10 yeast promoters from the pESC-Leu vector have the FLAG or
the MYC epitopes in the two MCS of the vector. Both include ATG codons at
the 5’ end of the FLAG and the MYC epitopes. If you clone into the Bgl II
or the Xho I site in frame with the ATG of the epitope tag, you will have
the start codon, kozak sequence and should get good expression of your N-
tagged protein. If you need the epitope tag at the C-terminal, you will
need to include your own start codon
This is the section from the manual that covers the same information.
The pESC vectors contain DNA sequences coding for epitope peptides that can
be specifically recognized by monoclonal antibodies. A sequence for the FLAG
® epitope (DYKDDDDK)1 is located in the multiple cloning site (MCS)
downstream of the GAL10 promoter; a sequence for the c-myc epitope (
EQKLISEEDL)2 is located in the MCS downstream of the GAL1 promoter. The gene
of interest can be inserted in front of the epitope sequence to generate C-
terminal tagging or after the epitope sequence for N terminal tagging. These
tags allow the protein of interest to be studied without generating a
specific antibody to that protein. The epitope tags can be easily detected
in transformed cells using well-characterized, commercially available
antibodies.
This is a link to the vector Circle Map that shows the information above.
http://www.genomics.agilent.com/files/Vectors/Maps/pdf/pESC-LEU |
h**********r
发帖数: 671 | 6 Addgene有这个的改进版，pesc-leu2d。
据说稳定性不错。
这个质粒其实是keasling lab改进的，把leu2的promoter缩短，弄得比较弱，从而拷贝
数变高了。碰到过文章的作者，他说挺好用的。
他们实验室后来很多工作都用这个系列的。
http://www.addgene.org/20120/ |
h**********r
发帖数: 671 | 7 另外你也可以考虑整合到基因组体上，反正yeast也好弄。 |
s******y
发帖数: 28562 | 8 谢谢！
or
at
II
【在 h**********r 的大作中提到】
: 必须是pESC系列。
: 当时我还问大家要不要加kozak序列，也没说出个所以然来。后来我终于等到了客服的
: 答复。下面就是：
: The GAL1 and GAL10 yeast promoters from the pESC-Leu vector have the FLAG or
: the MYC epitopes in the two MCS of the vector. Both include ATG codons at
: the 5’ end of the FLAG and the MYC epitopes. If you clone into the Bgl II
: or the Xho I site in frame with the ATG of the epitope tag, you will have
: the start codon, kozak sequence and should get good expression of your N-
: tagged protein. If you need the epitope tag at the C-terminal, you will
: need to include your own start codon
|
d******u
发帖数: 99 | 9 how about pBridge vector(clontech) |
j****n
发帖数: 3370 | 10 直接插入两个cassette 也不是很难
就是promoter1-gene1-terminator1-promoter2-gene2-terminator2
利用酵母自身homologous recombination克隆 很容易的
我们实验室经常一次性搞个5-10个cassette 也没太大问题
★ 发自iPhone App: ChineseWeb 7.8
【在 s******y 的大作中提到】
: 我们需要在酵母里同时高表达两个蛋白，但是不知道mamalian常用的那些IRES, P2A
: sequence这些东西能不能用在这种场合上？
: 我们本来想找两个抗性不一样的表达质粒各自表达一个，但是我们能找到的都是URA
: marker.这样会弄得很麻烦因为抗性没有差别。
|
i*****g
发帖数: 11893 | 11 有能bidirectional promoter yeast plasmid，多年不用，记不住了，
只是yeast 破壁比较麻烦，beat beater可以用，还可以用一个机器破壁 很方便
但机器也有好多种，其中一款非常好用，也不产生泡沫和变性，厂商忘记了，
有些机器不好用，明显产生泡沫，蛋白变性。 |
e***o
发帖数: 344 | 12 2A works in yeast according to some papers. I am also trying this. |
X******n
发帖数: 914 | 13 可以一个plasmid用Ura，一个用G419.
Addgene有susan Lindquist的一套yeast gateway vector，188个载体，好像800刀。
【在 j****n 的大作中提到】
: 直接插入两个cassette 也不是很难
: 就是promoter1-gene1-terminator1-promoter2-gene2-terminator2
: 利用酵母自身homologous recombination克隆 很容易的
: 我们实验室经常一次性搞个5-10个cassette 也没太大问题
:
: ★ 发自iPhone App: ChineseWeb 7.8
|
X******n
发帖数: 914 | 14 可以一个plasmid用Ura，一个用G419.
Addgene有susan Lindquist的一套yeast gateway vector，188个载体，好像800刀。
【在 j****n 的大作中提到】
: 直接插入两个cassette 也不是很难
: 就是promoter1-gene1-terminator1-promoter2-gene2-terminator2
: 利用酵母自身homologous recombination克隆 很容易的
: 我们实验室经常一次性搞个5-10个cassette 也没太大问题
:
: ★ 发自iPhone App: ChineseWeb 7.8
|
