Team:Wageningen UR/Notebook/Robert
Creating an Adhesion Protein Library
By: Robert Hooftman
In this notebook, the endeavors of Robert are described: the generation of an adhesion protein library. To learn about the results of this project, go to the Results page .
May
Week 1 (13th of May – 19th of May)
Got new office, all doing literature research and dividing subprojects.
Week 2 (20th of May – 26th of May)
Working on proposal and overall literature research. Visited BCF career event in Utrecht .
Week 3 (27th of May – 2nd of June)
Working on finding sponsors. Besides, doing literature research and writing proposal.
June
Week 4 (3rd of June – 9th of June)
Working on proposal, literature research and sponsor searching.
Week 5 (10th of June – 16th of June)
Finished proposal.
Week 6 (17th of June – 23rd of June)
Doing literature research into potential adhesion protein candidates and expression methods.
Week 7 (24th of June – 30th of June)
Identified 6 potential candidates. Looking for the sequences for the adhesion protein candidates.
July
Week 8 (1st of July – 7th of July)
Ordering gBlocks and primers. Working in lab, making stocks for media and cells. Amplifying backbone for protein production.
Week 9 (8th of July – 14th of July)
Amplification of potential adhesion protein candidates from Gblocks. Constructs were made and 3 out of 6 potential candidates were cloned successfully.
Week 10 (15th of July – 21st of July)
Sent 2 other candidates for sequencing, which were also correct. 5 out of 6 were correct. Ordered new primers for 6th candidate, since they appeared to be wrong.
Week 11 (22nd of July – 28th of July)
No lab work done due to absence.
Week 12 (29th of July – 4th of August)
6th candidate also successfully amplified and cloned into construct. Sequencing verified.
August
Week 13 (5th of August – 11th of August)
First attempt at protein production, using E. coli BL21DE3. One out of 6 (PD1764) worked.
Week 14 (12th of August – 18th of August)
Started first attempt at benchtop protein purification for the successful candidate (PD1764). Got no protein in elution fraction.
Week 15 (19th of August – 25th of August)
Second attempt at protein production. Again, one out of 6 worked. Second protein purification attempt for PD1764. Again, no protein present in elution fraction.
Week 16 (26th of August – 1st of September)
PD1764 is present in cell pellet in high volumes. It seems to have a N-terminal transmembrane region. Designing primers to amplify part without this transmembrane region.
September
Week 17 (2nd of September – 8th of September)
Making new version of PD1764: PD1764sh. Cloning this gene in plasmid, using Golden Gate. New attempt at protein production other candidates, but no success.
Week 18 (9th of September – 15th of September)
Cloning successful. Working on protein production for PD1764sh
Week 19 (16th of September – 22nd of September)
Successful protein production and isolation for PD1764sh using Strep gravity column.
Week 20 (23rd of September – 29th of September)
Ordered chitin magnetic beads to test chitin binding capacities. Working on E. coli surface display method for smaller variants. Ordered primers.
Week 21 (30th of September – 6th of October)
Chitin magnetic beads have arrived, doing first tests with them. Trying to find optimal conditions for the binding. Doing cloning for surface display method, using INPNC of X. campestris.
October
Week 22 (7th of October – 13th of October)
Sequencing showed failed display. Redid. Control GFP gave no visible fluorescence, so dropped. Do more test with chitin beads. Writing for results page.
Week 23 (14th of October – 20th of October)
Did new binding with PD1764sh with PD1764sh + BSA and dissolved chitin (4 combinations in total, triplicates), which gave a positive result. Writing for results page.