Trypanosoma Antigen Production

By: Linda van Oosten

Here you can find Linda's work on the production of antigens from various Trypanosoma species. Curious about her results? Read more about it here.

June

Week -3 until -1 (19th of June - 9th of July)
Contacted Philippe Büscher from the Institute of Tropical Medicine, Antwerp. He send me genomic DNA of three Trypanosoma brucei species.

July

Week 1 (10th of July - 16th of July)
Proposal writing and starting the in silico design. Talked to Philippe Büscher on the 14th of July for advice on protein expression.

Week 2 (24th of July - 30th of July)
Gave presentation in the Tuesday meeting, changed in silico design, ordered primers to amplify the gene for the extracellular domain of ISG64, ISG65 and ISG75 of Trypanosoma brucei gambiense flanked with KpnI and SacI restriction sites. Extracellular domains are found to be: ISG64 domain 24-365 ISG65 domain 21-385 ISG75 domain 28-468

August

Week 3 (31st of July - 6th of August)
Started on PCR’in the genes out of the genomic DNA. Both gambiense and rhodesiense species of Trypanosoma brucei were used in the PCR reactions, to see the specificity of the primers. Primers annealed for both species. Only the gambiense gene was used for further construct building. It was gel purified and digested with both KpnI and SacI. The backbone, pET52b, was obtained by miniprepping and was also digested with KpnI and SacI, present in the multiple cloning site of the plasmid. The digestion products were cleaned-up, and used for T4 ligation. Digestion of the plasmid could be confirmed on gel, however the ligation product could not be visualised on gel. Moreover, after transformation by heatshock to DH5alpha, no colonies formed. This week also, chemically competent E. coli Rosetta were made for future experiments.

Week 4 (7th of August - 13th of August)
The construct building was repeated, since it failed in week 3. First, the ligation was repeated using higher DNA concentrations. Once again, no colonies formed on the plates after transformation and the ligation could not be visualized on gel. Because of this, the construct building was done from scratch. Starting with PCR of the genomic DNA and continuing to the transformation. This time, DNA concentrations measured with Nanodrop were much better looking. Ligation was performed with 3:1 insert to vector, and could be visualised on gel, where both ligated product, excess insert and excess backbone (empty) were seen. Colonies on the plates after transformation were checked with cPCR. Most colonies seemed to contain the insert.

Week 5 (14th of August - 20th of August)
The colonies with the highest band intensities of week 4 were picked, grown overnight, miniprepped and sent for sequencing. Of 6 samples, 3 showed an empty backbone. The other three have the insert and are in-frame. However, some mismatches were observed comparing the sequencing data with the reference sequence obtained from online databases. Moreover, none of the pET52b-ISG75 constructs showed the insert inside the plasmid. More colonies of the transformation of week 4 were checked for the presence of the insert, but none were found.

Week 6 (21st of August - 27th of August)
Miniprepped and transformed ⅔ of all constructs to Rosetta strain. Sequencing original template, compared it to sequence of constructs, looked okay, mutations were already there. Re-made the ISG75 construct in DH5a, because if failed the first times, succeeded this time.

September

Week 7 (28th of August - 3rd of September)
Sequenced the ISG75 construct and original template, looked shitty so decided to drop that one. Miniprep and PCR - checked the other two constructs that were transformed into Rosetta. Tested protein expression (induction of promoter, and if the protein is soluble).

Week 8 (4th of September - 10th of September)
Checked protein expression on SDS, looked fine. Scaled up and did the protein expression and purification protocol.

Week 9 (11th of September - 17th of September)
Checked the purification with Nanodrop, ran it on a SDS-gel and did the protein quantitation assay. Expression of rISG65 seemed very low, checked the construct by sequencing to make sure there is no error in the sequence. Sequence looked fine. Ordered primers for BioBrick formation.

Week 10 (18th of September - 24th of September)
Started to make biobricks from my sequences. Did PCR to add the prefix and suffix to my insert, and cloned it into the linearized pSB1C3 backbone. Ligation was checked on gel. Colonies were checked with cPCR.

October

Week 11 (25th of September - 1st of October)
Colonies that looked fine on cPCR were grown overnight, miniprepped and sent for sequencing. The sequence was correct, hence I finished making the BioBricks.

Week 12 (2nd of October - 8th of October)
Performed the serum experiment to demonstrate that E. coli cells can survive in (horse) serum. Also started testing the BioBricks of TUDelft by transforming it to competent BL21(DE3).

Week 13 (9th of October - 15th of October)
Finished testing the BioBricks from TUDelft. Did another batch of ISG64 protein expression and purification. This time, the cell lysate was incubated together with the beads for one hour, and only part of the beads were eluted. The rest was stored at 4 degrees for later use.

Week 14 (16th of October - 22nd of October)
Re-did the expertiments from TUDelft with more duplicates. Results were similar to the first time. This week, the deadline for Human Practices on the Wiki took place, meaning most time was spent on Wiki stuff.

Week 15 (23rd of October - 29th of October)
Continued working on the Wiki. Did another fresh batch of ISG64 protein expression and binding to sepharose beads. Helped with the phage display selection, where these beads are used.

Team:Wageningen UR/Notebook/Journal/Trypanosoma