Team:Calgary/Notebook
NOTEBOOK
Below are notes regarding all CRISPR experiments.
May
Week 1
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Week 2
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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. 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 5
We received very generous samples of T-Rex Flp-In HEK293 cells and pcDNA5 and pOG44 vectors from Dr. Ray Wang, a researcher in Dr. Wayne Chen's lab here at the University of Calgary. The cells were stored at -80C while the pcDNA5 and pOG44 vectors were transformed into chemically competent DH5-α. We miniprepped several samples of pcDNA5 and pOG44 from the cells and have stored them in the freezer for use later in the summer. We also made glycerol stocks of both vectors for long-term storage.
We re-transformed all of the Interlab test parts in chemically competent DH5-α. During the transformation we used a lower volume of recovery media and lengthened the recovery incubation. All of the transformations grew much quicker than last week, therefore we will use the new, faster-growing parts for the remainder of the Interlab study.
Addgene was offering free synthesis of 2kb gene fragments. We took full advantage of this offer and ordered a few of our constructs: mCherry-BGH, which will be ligated to CMV (BBa_K747096) from the distribution kit, and both CHS4 insulator parts. Before CMV is ligated to mCherry-BGH, we will use overhang PCR to add a SalI restriction site upstream of the promoter. This will improve the part and allow it to function with our construct system.
Week 6
We began the week by testing electroporation protocols using our electrocompetent cells and the competent cell test kit. The first protocol that we tried was very successful, and we saw very high transformation efficiencies. This allowed us to conclude that electroporation will be the method of choice for transforming more important parts into DH5-α cells. We also created a plasmid stock of the vectors pcDNA5 and pOG44 for later use in HEK293 Flp-in T-REx cells. We tried to miniprep and digest confirm the remaining Interlab test parts, but continued to get unexpected results. Therefore, we re-transformed test parts 1, 5, and 6 (BBa_J364000, BBa_J364008, BBa_J364009). We designed PCR primers with overhangs to add restriction sites to CMV from the registry that will later be cloned with an mCherry-BGH construct to form our insert. We conducted more in depth research into methods and protocols for qPCR and bisulfite sequencing. Since IDT was unable to synthesize our UCOEs, we contacted Genscript and they provided us with a quote. Finally, we investigated using Gibson assembly as an alternative method for assembling our constructs.
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.