Team:Wageningen UR/Notebook/Santi
Yeast-based Assembly of phages
By:Santiago Castanedo Fontanillas
In Santi's notebook you can learn more about yeast-based phage engineering and other phage engineering methods to produce our Xylencer phage. His achievements can be found on this and this page.
May
Week 1 (13th of May – 19th of May)
Defined my subproject.
Week 2 (20th of May – 26th of May)
Defined my subproject.
Week 3 (27th of May – 2nd of June)
Defined my subproject.
June
Week 4 (3rd of June – 9th of June)
Defined my subproject.
Week 5 (10th of June – 16th of June)
Started writing my proposal.
Week 6 (17th of June – 23rd of June)
Kept writing my proposal.
Week 7 (24th of June – 30th of June)
Started in the lab.
Co-transformed the CEN/ARS + HIS3 regions of the pHLUM YAC plasmid and also a variant of the pBeloBAC11 plasmid, with regions of homology between them, into CENPK-1D yeast cells, to build my BAC-YAC plasmid.
July
Week 8 (1st of July – 7th of July)
Transformed the extracted BAC-YAC plasmids from the positive yeast colonies into E. coli K12 DH10β cells.
Week 9 (8th of July – 14th of July)
Out for a conference.
Week 10 (15th of July – 21st of July)
Vacation.
Week 11 (22nd of July – 28th of July)
Tried to amplify 8 kb fragments from our own purified lambda genome. It didn’t work.
Week 12 (29th of July – 4th of August)
Tried to amplify 8 kb fragments from a lambda genome that was purified using the phenol and chloroform method. Still no results.
August
Week 13 (5th of August – 11th of August)
Optimized the protocol for the isolation of lambda genome with the phenol and chloroform method by increasing the centrifugation speed and eliminating all residual ethanol. The amplification of 8 kb fragments was still unsuccessful.
PCR amplified all the DNA sequences required to construct the 8 plasmids necessary to test the phage capsid fusion protein expression. It was successful.
Week 14 (12th of August – 18th of August)
Successfully PCR amplified the gpd gene from our own isolated lambda phage genome.
Digested all the different PCR amplified DNA sequences to design the 8 plasmids. Ligation was then performed only for 3 of the inserts of the plasmids containing the ribosomal frameshifting sequence.
Transformed the BB_J23100 plasmid from the iGEM plates into E. coli cells. It yielded colonies.
Week 15 (19th of August – 25th of August)
Unsuccessfully amplified 8 kb fragments from our own purified lambda phage genome.
Unsuccessfully amplified the BAC-YAC plasmid using the Q5 polymerase. Purified again the BAC-YAC plasmid from a glycerol stock to repeat the experiment.
Week 16 (26th of August – 1st of September)
Amplified different sized DNA fragments from the BAC-YAC plasmid with both One-Taq and Q5 polymerases. It was only successful with the One-Taq polymerase and up to 5 kb fragments.
PCR amplified the 3 ligated inserts for the plasmids containing the ribosomal frameshifting sequence, and afterwards gel isolated the right bands.
Ligated the digested BB_J23100 plasmid with the respective inserts from the different 8 plasmids.
September
Week 17 (2nd of September – 8th of September)
Transformed the 8 plasmids into E. coli cells.
Due to the amplification of the BAC-YAC plasmid working with One-Taq polymerase, secondary structures in the DNA sequence might be the problem.
PCR amplified the BAC-YAC plasmid with the Q5 polymerase and DMSO, and with the Q5 polymerase with a One-Taq protocol. It worked better for the latter case.
Successfully PCR amplified 8 kb fragments from the newly bought phage lambda genome, when using Q5 polymerase with DMSO.
Week 18 (9th of September – 15th of September)
Transformed CENPK-1D cells with the 6 8 kb fragments amplified from the lambda phage genome and the linearized BAC-YAC plasmid. It was not successful.
Troubleshooting of the entire subproject: came to the realization that the cloning of the phage genome into the BAC-YAC plasmid would not work with the lambda phage, even though it works for the T7 phage.
Redesigned the subproject: added BsaI sites at the beginning and end of the BAC-YAC fragment.
Ran colony PCRs of the E. coli colonies transformed with each one of the 8 different plasmids used to test the phage capsid fusion protein expression. None were right.
Week 19 (16th of September – 22nd of September)
Transformed CENPK-1D cells with the 6 8 kb fragments amplified from the lambda phage genome, using the newly designed primers, and the linearized BAC-YAC plasmid. It was successful.
Re-ligated and transformed the plasmids containing the fusion gene construct with the ribosomal frameshifting sequence into E. coli cells. Colony PCRs were not successful.
The restriction – ligation approach to this part of the subproject was abandoned due to time constraints.
Week 20 (23rd of September – 29th of September)
Screened the assembled TAR plasmid (lambda phage genome plus the BAC-YAC sequence) by running colony PCRs of the yeast colonies, using primers that amplify different regions of fragment junctions. It was unsuccessful.
Week 21 (30th of September – 6th of October)
Further screened the assembled TAR plasmid with colony PCRs of the yeast colonies, using primers that amplify different regions of fragment junctions. Obtained possible candidates.
Did yeast plasmid purification of the TAR plasmids from the candidate colonies, digested with BsaI, and ligated the cut plasmids.
Transformed the ligated lambda phage plasmids into E. coli cells.
October
Week 22 (7th of October – 13th of October)
Cloned all the plasmids necessary for the engineering of lambda phage via homologous recombination using the help of the lambda red system.
Week 23 (14th of October – 20th of October)
Infected the cells from the bacterial strain containing the plasmid with the lambda phage capsid fusion gene and the plasmid with the lambda red system. Collected the phages.
Infected the cells from the bacterial strain containing the counterselection CRISPR-Cas system with the phages collected from the last step.
Plated the phages and screened for the correctly engineered phages. Performed chitin-binding assays with the engineered phages.
Designed the poster for the Giant Jamboree and designed figures for the presentation in Boston.