Plant gene expression in bacteria and yeast - (Apr/21/2005 )

IMPORTANT!!!PLEASE HELP!
1.Do I need a full ORF of a gene of interest to express it in bacteria or yeast???What if I have an ORF but not the whole ORF of a gene?Is there any sense to check its expression?
2. Can I use pCRII-TOPO for this expression?
3. Can I use pYES2 vector for expression in E. coli?
4. Can I use DH5Alfa strain for these expression?
5. And if I would like to express my gene of interest in pYES2 in S. cerevisiae or pBluescript(or pCRIITOPO) in E. coli, should I have its start and stop codon?
6. What if between restriction cloning site and Start codon of my gene of interest there are ~20 nucleotides, will that affect expression?
Sorry if you find these questions stupid or irritating, but I'm really interested in functional analysis of a plant gene in bacteria and yeast in order to check if it confers some stress and unfortunately I haven't got experience and knowledge in this subject, what's the saddest- I've got nobody who could help me:(
Thank you in advance!

1.Do I need a full ORF of a gene of interest to express it in bacteria or yeast???What if I have an ORF but not the whole ORF of a gene?Is there any sense to check its expression?

-> Let me try first!
for expression of a gene in both bacteria and yeast (low eukaryote) then you need a start and stop codon. For a bacteriai then the start codon can be ATG (for methionine) and for yeast then i think you need the same codon. For higher eukaryotes then you need Kozac sequence as the start codon. Stop codon can be one of the three codons which ones i can't remember. It is iokey if you don't add everything of your gene, but what is important is you have to have enough nucleotides so you don't affect the start and stop codon. Usually the vectors you use have added start and stop codon so you don't need to add them in your insert.

2. Can I use pCRII-TOPO for this expression?
->The vector you use must have expression signals that can be recognized by the host.

3. Can I use pYES2 vector for expression in E. coli?
->See above.

4. Can I use DH5Alfa strain for these expression?
-> i once used this E. coli strain for gene expression, but i don't know if you can use it.

5. And if I would like to express my gene of interest in pYES2 in S. cerevisiae or pBluescript(or pCRIITOPO) in E. coli, should I have its start and stop codon?
->if your vector have these sequences then you don't need to add to your insert, if they don't then you have to add in your insert.

6. What if between restriction cloning site and Start codon of my gene of interest there are ~20 nucleotides, will that affect expression?

->you have to count that every 3 nucleotides account for an amino accid, so you have to make sure that they don't get into your insert.


Please, correct me if i am wrong.

Lord! I must have been very sleepy. I answered without thinking that it is plant gene you talk about.

Dear JPJ,

Ur queries are answered here. Hope these will be of some use to u.

1.Do I need a full ORF of a gene of interest to express it in bacteria or yeast???What if I have an ORF but not the whole ORF of a gene?Is there any sense to check its expression?
REPLY : Its not necessary u need the full ORF to express ur gene in bacteria or yeast. You can use expression vectors with fusion tags to supplement whats lacking in ur gene of interest. Try pET32, pRSET, pGEX etc. pET32 is one of the easiest. U have N terminal and C terminal fusion tags in the vector. Just insert the gene in the MCs. doesnt matter if u dont have a stop codon or stop codon in ur insert. As long as ur gene is inserted in the right reading frame, u have a good chance of getting the right recombinant protein.

2. Can I use pCRII-TOPO for this expression?
REPLY : PCRII-TOPO is a cloning vector. It is not optimised for recombinant protein expression. Try pET, pRSET, pGEX vectors for expression in E.coli.

3. Can I use pYES2 vector for expression in E. coli?
This vector is meant for the inducible expression of recombinant proteins in S.cerevisiae. Yeast and bacterial expression vectors differ in their basic molecular biology. Its unlikely that this vector will be suitable for expression in bacteria.

4. Can I use DH5Alfa strain for these expression?
REPLY: You can use DH5 alpha for expression. (provided u use a compatible expression vector)

5. And if I would like to express my gene of interest in pYES2 in S. cerevisiae or pBluescript(or pCRIITOPO) in E. coli, should I have its start and stop codon?
REPLY : No need for the start and stop codons if u use a fusion vector like pET32. (ref to reply to question one for further details)

6. What if between restriction cloning site and Start codon of my gene of interest there are ~20 nucleotides, will that affect expression?
REPLY : Generally this wont affect. But please consider the following points
a) If u are using an N terminal fusion vector ( U use the start codon of the N terminal fusion protein, and ur protein comes later - please make sure that there are no STOP CODONS (TAA, TAG or TGA , in frame between ur gene and the N terminal fusion tag.
If u are using the start codon of the gene of ur interest, please ensure that ur start codon is not placed too far away from the ribosomal binding site (RBS) due to the presence of the intervening sequences..

regards
avinash

http://aviprem.gq.nu