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.
Week 7
We determined that the cloning method we will proceed with will be the traditional restriction enzyme cloning, while other members of the team may later pursue Gibson assembly if our method is not working. Having decided that we would PCR amplify the subpart of A2UCOE that constitutes CBX3-UCOE as a method for staying on budget, we ordered only A2UCOE from Genscript. We finished the CMV overhang-primer design on Monday and ordered them from the University of Calgary DNA services. We miniprepped pSB1C3-CMV that the CRISPR subgroup had previously transformed and digest confirmed, and then ran PCR to generate CMV with restriction sites that we will need. The product was run on a gel and appears to be the correct size, so we proceeded with a digest. Our mCherry-BGH and one of the insulators that had been ordered as free samples from Genscript arrived on Friday, and will be used early next week. For the Interlab, we miniprepped and digest confirmed the parts once more, and decided to proceed. We attended an orientation at the Snyder Core Facility, which is where the fluorescent plate reader that we will need to use for the Interlab study (as well as the qPCR machine) is located. Following more in depth training in the Snyder Core Facility, we will be able to complete the Interlab study. We helped train the CRISPR subgroup in using electroporation. Finally, we participated in HEK cell training, including passaging and transfecting cell cultures.
Week 8
This week, our ordered parts arrived and we began to clone them into pSB1C3. We digested our mCherry DNA block, the CMV PCR products with XbaI and PstI, and a previously miniprepped backbone sample which we then ligated. Simultaneously, we digested both the cHS4 insulator DNA blocks, as well as more backbone with XbaI and SpeI then ligated. After some ligation troubleshooting, we proceeded to transform chemically competent DH5-α cells with the ligation products as well as electroporate some samples. However, as of Friday these transformations were unsuccessful. Aside from the wet lab work, we collaborated with the dry lab team to discuss wiki content for our subgroup. In addition, we researched some protocols that will be used later this summer. Finally, we read the Thermofisher manuals for the T-REx HEK 293 cells that we will begin to use in the future.
Week 9
We began the week by screening colonies from the ligations of our parts into pSB1C3 using digest confirmations. The first colonies screened did not contain the correct inserts, but on the second attempt, we found some colonies that appeared to possibly contain our improved pSB1C3-CMV and pSB1C3-mCherry-BGH. These samples were sent for sequencing and we are awaiting the results. As we had issues with trying to ligate the cHS4 insulators into pSB1C3 using XbaI and SpeI sticky ends, we repeated the ligation using a 7:1 insert to vector ratio. The ligation products were transformed into chemically competent DH5-α, and we screened some of the new colonies. Unfortunately, all the results were negative. Outside of the lab, we completed the iGEM Safety Form 1, and began writing content and creating figures for our wiki pages.
July
Week 10
Early in the week, we completed a large portion of the Interlab study by generating the required standard curves. We received the sequencing results for the suspected improved pSB1C3-CMV and pSB1C3-mCherry-BGH, but the sequences were not as expected. It appeared that what we suspected to be the backbone with the correct insert was actually residual undigested backbone, even though we had gel-extracted it before ligation. These parts were digested and ligated once more.
After using digest confirmations to screen approximately 60 colonies in the first half of the week, we discovered a colony PCR (cPCR) protocol that would optimize and streamline our colony screening process. We tested this protocol using the positive control from the Interlab study (BBa_I20270) in order to determine which buffer and Taq polymerase combination works best. Having isolated the best combinations, we are confident implementing this solution moving forward.
Week 11
We began the week by repeating some transformations as well as digests and ligations of the cHS4 insulators and the improved CMV product into pSB1C3. In addition to trying to ligate the insulators into pSB1C3 using XbaI and SpeI sites (as the Genscript part is missing the NotI site from the prefix and suffix), we also attempted to use the EcoRI and PstI sites. Using colony PCR (cPCR), we were able to screen approximately 130 colonies with ease. We found one colony with pSB1C3-mCherry-BGH, and ran an addition digest confirmation as well as sent a sample for sequencing. We were also able to find four colonies for the E/P-digested pSB1C3-cHS4(1) and one for E/P-digested pSB1C3-cHS4(2). The ligation of improved CMV into pSB1C3 produced no colonies, but we moved ahead with ligating the improved CMV into pSB1C3-mCherry-BGH, and transforming the product. These colonies will be screened next week. In order to perform experiments to test the function of our CMV as an improved part, we also began steps to clone the original CMV promoter from the registry into pSB1C3-mCherry-BGH. Finally, as the E/P digest-ligations worked so much more efficiently than those with X/S, we began investigating alternative methods for correcting the NotI sites on the cHS4 insulators within the backbone.
Week 12
As the CRISPR subgroup was mostly absent this week, we assumed their task of cloning together the two halves of the multiple cloning site (MCS). IDT was unable to synthesize the MCS as one part due to hairpins. Early in the week, we worked on digesting and recovering each half for ligation. The MCS first half was successfully digest confirmed, sequenced, and set aside for ligation. However, after several failed digests and sequencing, we concluded that the CRISPR subgroup's 'MCS second half' glycerol stocks actually contained empty backbone. Before we can move forward with joining the two halves, we will need to ligate the IDT Biobrick MCS second half into pSB1C3.
Our pSB1C3-mCherry-BGH sequencing results came back early in the week, and the part is exactly as expected. We are confident moving forward with cloning. We continued to screen pSB1C3-mCherry-BGH + improved CMV ligation colonies using cPCR, but so far we have not found a colony with the correct insert. As all of our improved CMV ligations have been unsuccessful, we began research into possible causes. We found that impurity of PCR products may be a factor, and therefore tried to gel purify our product. However, our gel purification kit failed once again. Next week, we plan to make new improved CMV and excise the correct band from LMP gel before digesting and ligating.
Sequencing results for both cHS4 insulators ligated using E/P showed that the correct part was inserted, but as suspected, the NotI site was mutated. This was further verified by a digest confirmation. In order to submit these parts, we will have to follow a two-step cloning process beginning with XbaI and PstI digestions. Colonies were screened using cPCR. Over the course of the week, we found only three colonies for the pSB1C3-cHS4(2) X/P ligation that appeared to have the correct insert. These will be sequenced next week.
As we intend to submit our new CMV as an improvement on CMV (BBa_K747096) from the iGEM registry, we also worked on cloning this part into pSB1C3-mCherry-BGH. After experiments meant to quell some doubt regarding the identity of glycerol stocks made by the CRISPR subgroup, we were able to digest pSB1C3-CMV with XbaI and SpeI and recover the insert via LMP gel extraction. Ligation into pSB1C3-mCherry-BGH will occur next week.
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.