Team:Wageningen UR/Notebook/Journal/Affinity Library
Affinity Body Library and Phage Display
By: Jurre Steens
Here you can find Jurre's work on the affinity body library and the accomponying phage display he performed. Curious about his results? Read more about it here and here.
May
Week 1 (1st of May – 7th of May)Proposal writing.
Week 2 (8th of May – 14th of May)
Proposal writing.
Week 3 (15th of May – 21st of May)
In silico design.
Week 4 (22nd of May – 28th of May)
In silico design.
June
Week 5 (29th of May – 3rd of June)Making electrocompetent cells, getting used to the lab.
Week 6 (4th of June – 10th of June)
Growing plasmid backbones and miniprepping.
Week 7 (11th of June - 17th of June)
First PCR performed.
Week 8 (18th of June – 24th of June)
Making and testing more electrocompetent cells.
July
Week 9 (25th of June - 1st of July)Amplification of glycerol stock pComb3XSS backbone and performed accomponying restriction digestion analysis. Designing primers to remove unwanted BsaI site in backbone and ran PCR to obtain said backbone with removed BsaI site. PCR amplification of Helix 3 fragment.
Week 10 (2nd of July - 8th of July)
Preparation of all fragments for 3-part ligation assembly as well as transformation using this ligation mixture. No colonies, so repeated ligation.
Week 11 (9th of July - 15th of July)
After new transformations, no success was observed so switched the assembly strategy. Designed primers for new strategy, performed PCRs to obtain assembly fragments and ligated+transformed again. Colony PCR to obtain the correct colonies.
Week 12 (16th of July - 22nd of July)
From the colony PCR, the correct colonies were picked and grown. Backbone now has the insert, so next step is to remove unwanted BsaI site.
Week 13 (23rd of July - 29th of July)
Designed more primers and performed more PCRs. Parts were ligated and subsequently transformed. Colonies picked, grown and sent for sequencing. Annealed Helix 1 & Helix 2 oligos to prepare for insertion in backbone. Wildtype IgG affibody-expressing vector assembled.
August
Week 14 (30th of July - 4th of August)Digestion of backbone to ligate Helix 1 & Helix 2.
Week 15 (5th of August - 11th of August)
Repeated same stuff as in week 13, found out there was a mistake in one of the oligos used. Ordered a new one and repeated said stuff.
Week 16 (12th of August - 18th of August)
For phage amplification and phage display, a F pilus strain of E. coli has to be used. For this reason the XL1-Blue MRF’ strain was retrieved, which was already available in the -80 storage. Electrocompetent XL1-Blue MRF’ cells were made and helper phage amplification was performed.
Week 17 (19th of August - 25th of August)
New XL1-Blue cells were ordered, received and made competent. Wildtype-IgG affinity body expressed in M13 phage. Colony PCR to check for insert.
September
Week 18 (26th of August - 1st of September)Wildtype-IgG affinity body expressed in XL1-Blue cells. Colony PCR showed good results.
Week 19 (2nd of September - 8th of September)
Phages expressing wildtype-IgG affinity body production. Titrations were performed and new XL1-Blue cells were infected. Colony PCRs performed on infected XL1-Blue colonies. Infective titer determined.
Week 20 (9th of September - 15th of September)
Infected XL1-blue strain with Phage-wildtype-IgG sequenced.
Week 21 (16th of September - 22nd of September)
A lot of trial and error. Affinity body constructs are working. Ligations performed.
Week 22 (23rd of September - 29th of September)
Upscaling of ligation mixture. Transformations performed. Sequences showed empty backbones, unfortunately. One of the oligo’s used contained an error, so ordered a new one (again).
October
Week 23 (30th of September - 5th of October)All ligation steps performed again with new oligos. Transformed again. Sequencing showed good results this time. Three good colonies picked and used for recombinant phage production.
Week 24 (6th of October - 12th of October)
Infective titer of recombinant phages determined.
Week 25 (13th of October - 19th of October)
Upscaling of ligations. Shitton of transformations performed. 96 colony PCRs, cleanup and sequencing. Library bias analysis. Phage library production and setting up Phage display.
Week 26 (20th of October - 26th of October)
Started with phage display panning. Made a boatload of plates and performed an equal amount of colony PCRs. Results of phage display.
