Jump to content

Phage-display library preparation, how do we obtain phages that contain both the protein and the DNA that encodes for this protein?


Recommended Posts

Hi there, hope everyone is remaining safe and well!

I was looking into phage-display library and have a question regarding the production. As far as I understand, one produces random mutations in some way (sources I am reading use degenerate primers), then these mutated primers are ligated into phagemids (not necessary, but this is I suppose safer as there is a need for helper phages), then these phagemids are transformed into bacteria.
My question stems from the size of these libraries (being about 10^8 to 10^10), and the fact that each phage will contain the gene that encodes for that exact protein. So my question is, if we have our mixture of phagemids, each with different sequences, how do we make sure that each bacteria only contains one phagemid DNA particle (or at least, not several different ones). I imagine that if a bacteria contains two different phagemids, it could be possible that the phage displays a different protein than the DNA it contains. 

I thought, that we may just separately transform each bacteria, but due to the size of the library this seems unfeasible. I think my reasoning is going awry somewhere. Could anyone explain to me how we do this, or where the fault in my thinking lies?

Sources I have used: https://www.jbc.org/content/273/34/21769.full.html 

pDN332 is a derivative of phagemid pHEN1 (7), in which the sequence between the NotI site and the amber codon preceding the gene III has been replaced by the following sequence, coding for the D3SD2-FLAG-His6 tag (22). 

Transformations into TG1 Escherichia coli strain were performed according to Marks et al. (6), and phages were prepared according to standard protocols (9). Five clones were selected at random and sequenced to check for the absence of pervasive contamination. The percentage of clones that express folded antibodies was determined by immunoblot and dot-blot analysis using anti-FLAG M2 antibody (Eastman Kodak Co.) and anti-mouse horseradish peroxidase immunoglobulins (A2554; Sigma, Buchs, Switzerland) as detecting reagents or protein A-horseradish peroxidase as described (9). Protein A binds strongly to folded VH domains derived from the DP47 segment (23).

Reference 6 is https://www.sciencedirect.com/science/article/abs/pii/002228369190498U (behind paywall unfortunately, although here is a copy of what I think is the relevant bit): 

(d) Cloning of the XFI? gene repertoires Purified DNA of the scFv gene repertoires (1 to 4 pg) was digested with Not1 and either A’$1 or Xc01 restriction enzymes. (The 2 different restriction enzymes were tried in an attempt to increase the cloning efficiency.) After digestion, the fragments were extracted wibh phenol/ chloroform, and ligated into pHEN1 (Hoogenboom et al.. 1991) vector that had been digested with either &‘$I and lVotI or LVcoI and Not1 and electroeluted from a @So/o agarose gel (Sambrook et al.. 1990). Each scFv gene repertoire was combined in a ligation mixture which included 6 pg of digested vector. in a 100 /d ligation mix with 2000 units of phage T4 DNA ligase (New England Biolabs) overnight at room temperature. The ligation mix was purified by extraction with phenol and precipitation with ethanol. The ligated DNA was resuspended in 10 ~1 of water, and 2.5 ~1 samples were electroporated (Dower et al., 1988) into 50 pl Escherichia coli TGl (Gibson. 1984). Cells were grown in 1 ml of SOC (Sambrook et al., 1990) for 1 h and then plated on TYE (Miller. 1972) medium with 100 pg ampicillin/ml and 1% (w/v) glucose (TYEAMP-GLC), in 243 mm x 243 mm dishes (Nunc). Colonies were scraped off the plates into 10 ml of 2 x TY broth (Miller, 1972) containing 100 pg ampirillin/ml. 1:/b
 

the first paper also refers to this paper (reference 9), although I don't think it is as relevant:
https://www.embopress.org/doi/epdf/10.1002/j.1460-2075.1994.tb06308.x

Thanks in advance
-Dagl

Edit:
Watching a video of Creative Biolabs, they mention that the phage display library is produced from 10^8th independent E. coli transformations. How? I presume this is done with some machine, because I cannot imagine some poor souls transforming 10^8 different wells? 

 

Edited by Dagl1
Found more information
Link to comment
Share on other sites

Create an account or sign in to comment

You need to be a member in order to leave a comment

Create an account

Sign up for a new account in our community. It's easy!

Register a new account

Sign in

Already have an account? Sign in here.

Sign In Now
×
×
  • Create New...

Important Information

We have placed cookies on your device to help make this website better. You can adjust your cookie settings, otherwise we'll assume you're okay to continue.