Team:Calgary/Notebook
NOTEBOOK
Below are notes regarding all CRISPR experiments.
May
Week 1
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Week 2
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas vitae feugiat odio. Morbi luctus lacus in suscipit ultrices. Suspendisse quis ultricies sapien. Vestibulum vitae massa eu dolor cursus aliquam vitae eget neque. Mauris a turpis nec mauris luctus blandit. Nunc suscipit, nisl nec fringilla placerat, magna nisi imperdiet est, nec interdum quam nulla nec quam. Nunc ligula dolor, convallis quis placerat eget, dapibus ut diam. In quis iaculis magna, a tristique magna.
Below are notes regarding all CME (chromatin modifying elements) experiments.
May
Week 1
We began an extensive literature search on the topic of insulators and other epigenetic regulatory elements. We determined that ubiquitous chromatin remodeling elements (UCOEs) would be effective at opening chromatin and will be very useful in producing continuous high-level transcription of any transgenes that are inserted into the human cell genome via our CRISPR/FLP system. While UCOEs will allow for our gene to enter an active transcriptional state, insulators will function to maintain this state. If flanking the transgene, insulators protect the gene from the spread of neighboring heterochromatic regions and from crosstalk of enhancers. In addition, we began a discussion about the testing platform that we will utilize. Working in parallel with the CRISPR subgroup prevents us from using their integration scheme, so we are investigating other options for preliminary experiments. We also completed all necessary Lab Safety training which will allow us to work in the lab this summer.
Week 2
We continued with literature research specifically focused on ubiquitous chromatin remodeling elements (UCOEs) as well as insulators. The A2UCOE, CBX3-UCOE, and cSH4 insulator were selected for experimentation and their sequences were obtained. We began planning constructs which will eventually constitute the various cassettes during recombinase mediated cassette exchange (RMCE) procedures. We continued the investigation of other options for preliminary experiments that will free us from dependence on the CRISPR subgroup's FRT integration scheme. One promising approach would be to generate minicircles from triple-FRT containing FLP plasmids within prokaryotic cells, isolate and purify the minicircles, and co-transfect them with plasmids expressing FlpO (pCAG-FLPo) into a HEK cell line that already contain one FRT site, such as the Flp-In T-REx HEK293 cells available commercially. This would allow for the insertion of a second heterospecific FRT site, which makes RMCE possible. Therefore, we could conduct our experiments on UCOEs and insulators while the CRISPR subgroup fine-tunes the CRISPR-mediated FRT integration scheme. We began a discussion about the process of obtaining the Flp-In T-REx HEK293 cells from internal contacts within the University of Calgary. Another promising possibility explored is to obtain a cell line that already contains heterospecific FRT sites. In this case, we could begin immediately with RMCE in order to investigate UCOE and insulator function. We contacted the principle investigator from that research group in regards to obtaining cells from the generated cell line and are awaiting a response.
Week 3
This week, we finalized our BioBrick plans including the restriction sites to be used. Further research on the characteristics of these restriction sites was also conducted. After meeting with Dr. Ray Wang, we secured access to the Flp-In T-REx HEK293 cell line as well as the commercial vectors pcDNA5 and pOG44 designed for use in the system. Dr. Ray Wang also offered to provide troubleshooting assistance to the team when using this system. We practiced laboratory techniques by transforming chemically competent DH5-α cells with a CMV promoter (BBa_I712004) and a BGH terminator (BBa_K1150012), and chloramphenicol resistance (BBa_K143064). The University of Calgary also hosted a methods day, where we were able to attend presentations and speak to other researchers regarding certain laboratory techniques and protocols. In particular, we learned more about bisulfite sequencing, transfection techniques, and qPCR primer design. Our team also attended a welcome event for summer students, where we went bowling as a team-building activity. Finally, we helped to prepare slides and script for a presentation that will be given to mentors, industry professionals, and other interested general public attendees on May 23rd.
Week 4
This week, we continued working on our transformed DH5-alpha cells for practice, including creating overnights, mini-prepping, running a digest, and a digest confirmation. In addition, we created approximately 70 aliquots of chemically competent DH5-alpha cells for later use. We transformed the parts required for Interlab into our chemically competent DH5-α cells and left them to grow over the weekend. Growth was slow, therefore we will re-transform the Interlab parts next week with an improved transformation protocol that will hopefully result in a normal growth rate.
We finalized the restriction sites that will be used in collaboration with the CRISPR subgroup to ensure that all parts are compatible, and that we are ordering sequences in the most efficient way possible. We transformed parts from the registry (BBa_I1466, BBa_K316003, and BBa_K316003) that were selected for the restriction sites they contain, as they will be used to test the function of restriction enzymes that have been in our freezer for some time. In addition to participating in the Innovate Calgary workshop, we also contributed to a team presentation in which we revealed our project idea and experimental plan to faculty experts, mentors, and members of the public. This event was very useful as we were able to identify some strengths and weaknesses of the project and plan as well as other general feedback from the audience.
June
Week 1
We began an extensive literature search on the topic of insulators and other epigenetic regulatory elements. We determined that ubiquitous chromatin remodeling elements (UCOEs) would be effective at opening chromatin and will be very useful in producing continuous high-level transcription of any transgenes that are inserted into the human cell genome via our CRISPR/FLP system. While UCOEs will allow for our gene to enter an active transcriptional state, insulators will function to maintain this state. If flanking the transgene, insulators protect the gene from the spread of neighboring heterochromatic regions and from crosstalk of enhancers. In addition, we began a discussion about the testing platform that we will utilize. Working in parallel with the CRISPR subgroup prevents us from using their integration scheme, so we are investigating other options for preliminary experiments. We also completed all necessary Lab Safety training which will allow us to work in the lab this summer.
Below are notes regarding all dry lab activities.
May
Week 1
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas vitae feugiat odio. Morbi luctus lacus in suscipit ultrices. Suspendisse quis ultricies sapien. Vestibulum vitae massa eu dolor cursus aliquam vitae eget neque. Mauris a turpis nec mauris luctus blandit. Nunc suscipit, nisl nec fringilla placerat, magna nisi imperdiet est, nec interdum quam nulla nec quam. Nunc ligula dolor, convallis quis placerat eget, dapibus ut diam. In quis iaculis magna, a tristique magna.
Week 2
Lorem ipsum dolor sit amet, consectetur adipiscing elit. Maecenas vitae feugiat odio. Morbi luctus lacus in suscipit ultrices. Suspendisse quis ultricies sapien. Vestibulum vitae massa eu dolor cursus aliquam vitae eget neque. Mauris a turpis nec mauris luctus blandit. Nunc suscipit, nisl nec fringilla placerat, magna nisi imperdiet est, nec interdum quam nulla nec quam. Nunc ligula dolor, convallis quis placerat eget, dapibus ut diam. In quis iaculis magna, a tristique magna.