Team:Uppsala/Experiments
Lab preparation protocols
During the two first weeks of summer the team prepared all upcoming lab work. All necessary solutions were prepared, such as LB, SOB, and agar plates with different antibiotics. The team also got the opportunity to practice basic lab techniques under aseptic conditions, which included creating competent cells, streaking, and re-streaking agar plates. More details on how we worked each week can be found on the Notebook page. Down below, protocols of all the lab preparation work can be found.
Biobrick assembly protocols
The primers arrived and the team started with the biobrick assembly process. The goal was to create seven different versions of bovine FGF2. Together with our PI, Professor Anthony Forster, and our TA, Konrad Gras, different experiments were designed to create these versions. We cannot thank Forster and Gras enough for their help with guiding us through these experiments. More details of the parts we created can be found on the Parts page. Down below, protocols of all the biobrick assembly work can be found.
Small scale purification protocols
When these seven different versions of bovine FGF2 had been created, we had to purify them. First we decided to do it at a small scale in the lab. Here we could troubleshoot and adjust the process for when we were doing an upscaled purification. For more information of the purification results, see the Results page. Down below, protocols of all the small scale purification work can be found.
Large scale purification protocols
The team also did a large scale purification at Cytiva Testa Center AB. The team successfully upscaled the purification process of the bovine FGF2 wild type protein. We want to thank Cytiva Testa Center AB for giving us the chance to upscale our project. Thanks to all of the employees and experts working at Cytiva for helping us design the experiments and purify the protein. A big thank you to Chandra Sekhar Mandava for helping us with the Testa Center part of our project. All protocols followed at Cytiva Testa Center AB can be found down below.
References
[1] Liljeruhm, J., Gullberg, E., & Forster, A. C. (2014). “Synthetic biology: a lab manual”
[2] Thermo Fisher Scientific. (2012) “Thermo Scientific GeneJET Plasmid Miniprep Kit K0502, K0503” https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0012655_GeneJET_Plasmid_Miniprep_UG.pdf (Accessed Sep. 22, 2021)
[3] Thermo Fisher Scientific. (2013) “Thermo Scientific DreamTaq DNA Polymerase” https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012037_DreamTaqDNAPolymerase_5x500U_UG.pdf (Accessed Sep. 22, 2021)
[4] Thermo Fisher Scientific. (2015) “Thermo ScientificGeneJET PCR Purification Kit #K0701, #K0702” https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0012662_GeneJET_PCR_Purification_UG.pdf (Accessed Sep. 22, 2021)
[5] Eurofins Genomics. (2021) “Mix2Seq Sequncing Kit” https://eurofinsgenomics.eu/media/1587665/tubesequencing_flyer_dinlang_8seiter_wickelfalz.pdf (Accessed Sep. 22, 2021)
[6] New England Bioloabs (2021) “Double Digest” https://international.neb.com/tools-and-resources/usage-guidelines/double-digests (Accessed Sep. 22, 2021)
[7] New England Bioloabs (2021) “Ligation Protocol with T4 DNA Ligase (M0202)” https://international.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202 (Accessed Sep. 22, 2021)
[8] Bio-Rad Laboratories (2021) “Quick Start™ Bradford Protein Assay” https://kirschner.med.harvard.edu/files/protocols/BioRad_proteinassay.pdf (Accessed Sep. 22, 2021)
[9] Protocols were created together with experts at Cytiva Testa Center AB. https://testacenter.com/
