3 replies to this topic

[Leonid]

Hi Folks,

Recently, I would like to clone 4 genes into one expression vectors under control of one promoter in the vector, but the problem is one of gene (gene1) have opposite transcriptional orientation with others. I could clone the 3 genes together into vector first, but I'm not sure if I should put the upstream spacer of gene 1, which may include original promoter and SD sequence, into the vector with gene1. I mean should I add the any upstream sequence of gene1 or I just simply clone the ORF of gene1 behind the three genes into the vector.

Does Any body have experience for that?

Thanks

[Quasimondo]

Leonid, on Jan 28 2010, 02:03 PM, said:

Hi Folks,

Recently, I would like to clone 4 genes into one expression vectors under control of one promoter in the vector, but the problem is one of gene (gene1) have opposite transcriptional orientation with others. I could clone the 3 genes together into vector first, but I'm not sure if I should put the upstream spacer of gene 1, which may include original promoter and SD sequence, into the vector with gene1. I mean should I add the any upstream sequence of gene1 or I just simply clone the ORF of gene1 behind the three genes into the vector.

Does Any body have experience for that?

Thanks

Just clone by the 5' - 3' direction of this gene (start from ATG), add the ribosome binding sites upstream the coding region with the spacer about 3-11 nucleotide (in E. coli, normally I use the AGGAGG sequence). For example: NNNNNN (RE recognition site) + AGGAGG (RBS or Shine- Dalgarno sequence) + spacer + ATG ... (coding sequence). Maybe you can add more nucleotide near the cleavage site for the efficient digestion (reference: http://www.neb.com/nebecomm/tech_reference...ucleotides.asp)

[Leonid]

Thank you very much! Coud I use the same SD and space sequence in the vector or I have use different one? another question is what's recognition site?

Thanks

Leonid

Quasimondo, on Jan 28 2010, 09:06 AM, said:

Leonid, on Jan 28 2010, 02:03 PM, said:

Hi Folks,

Recently, I would like to clone 4 genes into one expression vectors under control of one promoter in the vector, but the problem is one of gene (gene1) have opposite transcriptional orientation with others. I could clone the 3 genes together into vector first, but I'm not sure if I should put the upstream spacer of gene 1, which may include original promoter and SD sequence, into the vector with gene1. I mean should I add the any upstream sequence of gene1 or I just simply clone the ORF of gene1 behind the three genes into the vector.

Does Any body have experience for that?

Thanks

Just clone by the 5' - 3' direction of this gene (start from ATG), add the ribosome binding sites upstream the coding region with the spacer about 3-11 nucleotide (in E. coli, normally I use the AGGAGG sequence). For example: NNNNNN (RE recognition site) + AGGAGG (RBS or Shine- Dalgarno sequence) + spacer + ATG ... (coding sequence). Maybe you can add more nucleotide near the cleavage site for the efficient digestion (reference: http://www.neb.com/nebecomm/tech_reference...ucleotides.asp)

[Quasimondo]

Leonid, on Jan 29 2010, 03:55 AM, said:

Thank you very much! Coud I use the same SD and space sequence in the vector or I have use different one? another question is what's recognition site?

Thanks

Leonid

Quasimondo, on Jan 28 2010, 09:06 AM, said:

Leonid, on Jan 28 2010, 02:03 PM, said:

Hi Folks,

Recently, I would like to clone 4 genes into one expression vectors under control of one promoter in the vector, but the problem is one of gene (gene1) have opposite transcriptional orientation with others. I could clone the 3 genes together into vector first, but I'm not sure if I should put the upstream spacer of gene 1, which may include original promoter and SD sequence, into the vector with gene1. I mean should I add the any upstream sequence of gene1 or I just simply clone the ORF of gene1 behind the three genes into the vector.

Does Any body have experience for that?

Thanks

Just clone by the 5' - 3' direction of this gene (start from ATG), add the ribosome binding sites upstream the coding region with the spacer about 3-11 nucleotide (in E. coli, normally I use the AGGAGG sequence). For example: NNNNNN (RE recognition site) + AGGAGG (RBS or Shine- Dalgarno sequence) + spacer + ATG ... (coding sequence). Maybe you can add more nucleotide near the cleavage site for the efficient digestion (reference: http://www.neb.com/nebecomm/tech_reference...ucleotides.asp)

Yes, you can do that. Also, the RBS sequence can affect the expression level of this gene, so choose carefully if you want to manipulate that (the one I suggest is the strong one, you can find in literature). The recognition site of the restriction enzyme you will use for ligating is what I mention (such as GAATTC in the case of EcoRI). To make my answer clearer, for example your genes orientation is like this: [promoter 1](RBS)-->(RBS)--->(RBS)---> <----(RBS) [promoter 2 (reverse orientation)], I will make it like this: [promoter 1](RBS) --->(RBS)--->(RBS)--->(RBS)---> or [promoter 1](RBS)--->(RBS)---> [promoter 2 (same orientation as 1)](RBS)--->(RBS)--->