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
how to express the gene in opposite transcriptional direction
Started by Leonid, Jan 27 2010 09:03 PM
3 replies to this topic
#1
Posted 27 January 2010 - 09:03 PM
#2
Posted 28 January 2010 - 08:06 AM
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
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)
#3
Posted 28 January 2010 - 10:55 AM
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
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)
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
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)
#4
Posted 28 January 2010 - 08:22 PM
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
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)
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
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)--->













