12 replies to this topic

[Biogareth]

I tried to perform one amino acid saturation library based on the protocol which you published in 2008, but it always failed because the first base has low diversity (see the picture below).  I tried to use different primers and annealing temperature, but didn’t work out either. Do you think probably the first position is biased to “G” (wide type), and is not possible to be mutated.
Every suggestion is welcome!

Attached Thumbnails

• 

[phage434]

Is this sequencing of a mixed population of viable cells? If this is from a cell based library, there could be strong selection for genes having an initial G in that position, perhaps because it is an essential AA of an enzyme site in a critical enzyme.  If this is just sequencing of a DNA construct before cell insertion, then your technique/protocol needs improvement.

[Biogareth]

phage434, on 28 July 2011 - 06:05 AM, said:

Is this sequencing of a mixed population of viable cells? If this is from a cell based library, there could be strong selection for genes having an initial G in that position, perhaps because it is an essential AA of an enzyme site in a critical enzyme.  If this is just sequencing of a DNA construct before cell insertion, then your technique/protocol needs improvement.

Hi phage434,
Thanks for your reply!
For my case, this library is cell-based library. As I know, it is normal that there are some base biases. But for my case, only the first base was stick to "G", and other next two positions are quite equally distributed.
Do you think I have to check the DNA construct first?

[phage434]

I'm still not clear on what you have done.  Have you done mutation (how?) with degenerate primers at that location, and then transformed cells?  What exactly was sequenced?

[Biogareth]

phage434, on 28 July 2011 - 01:22 PM, said:

I'm still not clear on what you have done.  Have you done mutation (how?) with degenerate primers at that location, and then transformed cells?  What exactly was sequenced?

Hi phage434,

I have mutated 3 bases (one amino acid) with degenerated primer (NNK) by two stages of PCR. PCR products were digested by DpnI, and then transformed into Dam+ E.coli cells. Inoculate around 100 ul transfomants into LB medium for incubation of overnight. Extract the plamid mixture from overnight culture by Mininprep. So I used this plasmid for sequencing.

[phage434]

Well, I think it is clear that your mutagenesis with your degenerate oligos worked, and that you were able to transform those into your cells.  But there is clearly selection happening in the outgrowth of the cell population, which selects for a G in that position.  As I mentioned initially, this could be because that specific residue is required for activity of the enzyme you are modifying.  You could perhaps make this clearer if you were to sequence a number of single colony isolates, and determine exactly what AA was present in each one.  What's the enzyme, and is it integrated into the chromosome, or is it on a plasmid?  Is there a chromosomal knockout of the native enzyme?

Another possibility (less likely) is that your oligo has low degeneracy.  You could try ordering a set of three oligos with A, C, T in that position and mix them (or not).  Mixed oligos in synthesis don't always come out as equal mixtures, and this might be a particularly bad example.

[Biogareth]

phage434, on 29 July 2011 - 05:04 AM, said:

Well, I think it is clear that your mutagenesis with your degenerate oligos worked, and that you were able to transform those into your cells.  But there is clearly selection happening in the outgrowth of the cell population, which selects for a G in that position.  As I mentioned initially, this could be because that specific residue is required for activity of the enzyme you are modifying.  You could perhaps make this clearer if you were to sequence a number of single colony isolates, and determine exactly what AA was present in each one.  What's the enzyme, and is it integrated into the chromosome, or is it on a plasmid?  Is there a chromosomal knockout of the native enzyme?

Another possibility (less likely) is that your oligo has low degeneracy.  You could try ordering a set of three oligos with A, C, T in that position and mix them (or not).  Mixed oligos in synthesis don't always come out as equal mixtures, and this might be a particularly bad example.

Hello Phage434,

Thank you for your nice explanation.
Actually, the target enzyme is on a plasmid, and this enzyme is not present in host cells (E.coli). I also performed another saturation library with the same enzyme, and work well. As the explanation you mentioned intially, I cannot agree with it, because even the first base "G" was fixed, there are still some amino acids will be translated (the other two bases are equally distributed with bases).
For the low degeneracy of oligos, I also considered it, and reordered the degenerated primer, and Sigma promised me that it is equally distributed, but the result is still similar, even worse at the other two bases sometimes.

[phage434]

Another possibility is that the presence of any except a few amino acids in that location produces a toxic protein product, selecting against most mutations, except for a few having specific AAs in that position.  Again, sequencing for some individual colonies would make that clear. Proline, for example, would interrupt alpha helices, and might be selected for in that position if the helix produced a toxic protein. (Wrong codons, of course, but you get the idea).

[Biogareth]

phage434, on 29 July 2011 - 11:09 AM, said:

Another possibility is that the presence of any except a few amino acids in that location produces a toxic protein product, selecting against most mutations, except for a few having specific AAs in that position.  Again, sequencing for some individual colonies would make that clear. Proline, for example, would interrupt alpha helices, and might be selected for in that position if the helix produced a toxic protein. (Wrong codons, of course, but you get the idea).

It's a good idea actually, thanks again.
Because the plasmid with target gene contains constitutive promoter, so if I change the constitutive promoter to tye inducer promoter, do you think we can avoid to produce the toxic protein, and still keep intact diverse DNA library?

[phage434]

Yes, and the best would be a T7 promoter which will not be active in the cloning strain at all.  What is the native AA in that position?

[Biogareth]

phage434, on 30 July 2011 - 06:02 AM, said:

Yes, and the best would be a T7 promoter which will not be active in the cloning strain at all.  What is the native AA in that position?

Alanine

[phage434]

So, perhaps 50% of your mutants still have an alanine in that position; a large fraction of the remainder have a glycine.  Are you sure you are removing unmodified template from your reactions? Carryover of the unmutated DNA would explain a lot of what you are seeing.  If this is being done with PCR, then perhaps you are using too much template.  You could also perhaps remove template by DpnI digestion following PCR amplification and prior to cloning.

[Biogareth]

phage434, on 30 July 2011 - 03:19 PM, said:

So, perhaps 50% of your mutants still have an alanine in that position; a large fraction of the remainder have a glycine.  Are you sure you are removing unmodified template from your reactions? Carryover of the unmutated DNA would explain a lot of what you are seeing.  If this is being done with PCR, then perhaps you are using too much template.  You could also perhaps remove template by DpnI digestion following PCR amplification and prior to cloning.

Yes, DpnI digestion step is always performed after PCR amplificaiton. Most of unmodified template is supposed to be removed by this step. I don't think it contains quite a lot of unmutated DNA, otherwise the other two bases should be dominately by wild-type bases (but in my case, both positions are equally distributed).