Difference between revisions of "Team:UC Davis/Notebook"

Safety Training to work with Mammalian Cell Cultures. This week we read literature on Mammalian Biosensors to develop a frame work for our Project.

Before visiting the Yurok Tribe we did background research on the community and the area. This included not only scientific papers on the compounds in the area, but also the legislative and cultural context. We began to seriously consider how best to integrate human practices into the design of our project.

This week the team spent three days on the Yurok Reservation in Klamath, learning about the concerns of the tribe and the history of the land. We were able to make connections with the Superfund research core that has been working with the tribe and has collected data concerning the contamination in the Klamath river basin.

We selected our reporter gene. After researching several options including luciferase, we selected EGFP. We acquired a plasmid already containing EGFP, which was coupled to the CMV strong constitutive promoter. We also made progress in choosing promoters that are related to the pathways we are interested in. We conducted research concerning physiological stress pathways. In particular, we studied literature published on the heat shock family of proteins. We began collecting FASTA files of our promoters of interest.

We continued to do research on stress pathways and begin reading on mammalian cell culture and compiled papers to reference later in our protocols. We also continued to learn how to use the HTML language, in order to code our wiki.

To kickoff wet lab we began our InterLab study measurements. Our final data and findings are reproduced on our InterLab wiki. We followed the supplied iGEM interlab study protocol, available here: https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf We required several attempts to successfully transform the required DNA into our bacteria and obtained a new source of competent cells for use in our later experiments.

High school students in the Innovation & Entrepreneurship Pre-College program at UC Davis came to visit our lab to learn about the biotechnology field and try hands-on lab work with bacteria. We documented this on our wiki. For this activity, we taught the high school students how to perform a heat shock transformation.

We continue to do research and begin to put up our wiki. We met with an environmental toxicologist, Michael Dennison, who has experience in making bioassays and he reviewed our design. We selected our promoters of interest: MT1, MT2 full, MT2 59 bp, MT2 60 bp version 1, MT2 60 bp version 2, GADD45a, GADD153, FGF-21, and CYP1a1. We obtained FASTA files from the published literature and began to design the promoters which were able to be synthesized by IDT. We also designed primers for all of our constructs from IDT. Work continued on presentation.

After completing the Interlab Study, we prepared our plasmid vector containing EGFP, our reporter gene.

The high school students in the Innovation & Entrepreneurship Pre-College program at UC Davis returned to see the result of their experiment and get a debriefing on what happened.

We continued designing primers and the promoters which we could have synthesized. Three of our promoters were unable to be synthesized, CYP1a1, GADD153, and MT2 full. These we planned to amplify out of genomic DNA of mammalian cells. For more detail concerning dry lab from this week, please download our Project Design Notebook, where we included the majority of our dry lab work. Work continued on presentation. This document is available on our Design page: https://2018.igem.org/Team:UC_Davis/Design

After obtaining our vector plasmid, pcDNA3-EGFP, we grew it up in large quantities using DH5a. We then miniprepped it out. The miniprep protocol we followed is available here: https://static.igem.org/mediawiki/2015/1/18/NUDT_CHIAN-Protoco1.pdf

We digested the purified plasmid using two restriction enzymes, BglII and NotI, following this protocol available from NEB: https://www.neb.com/protocols/2018/07/30/restriction-digest-protocol

Digests were checked using gel electrophoresis. We used a gel concentration of 0.5 grams of agarose in 50 mL of TAE buffer, and ran it at 120 volts for 15 to 35 minutes, depending on the specific sample. Gels were then imaged on a Biorad ultraviolet gel imager. We used Sybersafe as our indicator, rather than ethidium bromide for safety considerations.

This week we continued to develop our wiki. After selecting our stress pathways of interest and promoters, we shifted our research focus to mammalian cells, promoters, and chemicals of concern. From our research we began to create own our protocol for culturing mammalian cells. To view our work from this week of research, please visit our project design notebook, where we documented the majority of our dry lab and literature review. To view our protocol for mammalian cell culture, please visit our Experiments page. Work continued on presentation.

We began to obtain materials for our mammalian cell culture. We also set up our dewer and biosafety cabinet. Work continued preparing our vector plasmid. After our restriction digest, we tested the quality of our digest by performing a negative control ligation, followed by a heat shock transformation into DH5a. Initial samples were of poor quality so we repeated the previous protocols until we had a satisfactory supply of restriction digested vector.

We amplified our synthesized promoters using PCR. We followed a PCR protocol for the Q5 polymerase, available from NEB

After PCR amplification, we purified our PCR products using a silica column.

After purification, we quantified the amount of DNA present in our samples using a Nanodrop machine from Thermofisher.

Our mammalian cell culture protocol was finished and we began to write our measurement protocol. Both finished protocols are available at our Experiments page, https://2018.igem.org/Team:UC_Davis/Experiments . Work continued on presentation.

We began to culture mammalian cells, following our prepared mammalian cell culture protocol.

For three of our constructs, MT2_2, MT2_3 and MT2_4, we annealed our synthesized oligos together and performed a SLIC T4 Polymerase chewback reaction on our digested plasmid vector. A copy of this protocol is available here: https://aem.asm.org/content/aem/suppl/2012/07/06/AEM.00844-12.DCSupplemental/zam999103465so2.pdf

We began to assemble our promoter-reporter gene constructs. We followed the following T4 ligase protocol, available from NEB, to assemble our restriction digested DNA: https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202

After ligation, we followed the above provided heat shock protocol.

We confirmed our transformed colonies by Colony PCR. For colony pcr we used Taq polymerase, following this protocol: https://www.neb.com/protocols/1/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273

We continued to do more research on measurement as well as transfection of mammalian cells. We continued work on our wiki. Work continued on presentation.

Mammalian cell culturing and viability testing continues. We continue to work on our constructs as we grow up more CHO and AML cells. After initial colonies for several constructs failed colony PCR, we repeated the entire workflow. After constructs passed colony PCR verification, we prepared samples for sanger sequencing and sent them to a local sequencing service: https://genewiz.com.

We researched relevant concentrations for our chemicals of concern. We analyzed sanger sequences from genewiz for our submitted samples. Work continued on presentation.

Mammalian cell culturing and viability testing continues. Constructs which were confirmed by Sanger sequencing were grown up in DH5a. Glycerol freezer stocks were made. Plasmid was isolated by miniprep. Plasmid concentration was measured by nanodrop and concentrated using a Speedvac. Samples were loaded, temperature set to 55º C and spun until the desired volume of water had been removed from the sample. We conducted additional colony PCR screening for constructs which failed sanger sequencing. We also repeated the entire workflow for constructs which failed additional colony PCR screening.

Work continued on protocols and the wiki. Work began assembling what we learned about human practices into a comprehensive document. Work continued on presentation.

Mammalian cell culturing and viability testing continues. We repeated the entire workflow for constructs which failed previous colony PCR screening. We then tested our new constructs using colony PCR. Colony PCR verified constructs were prepared for sanger sequencing. Genomic DNA was extracted from AML-12 cells and CHO-DG44 cells. We attempted to amplify out our promoters, CYP1a1 and GADD153 from this genomic DNA. The CYP1a1 amplification failed for unspecified reasons, possibly due to errors in primer design.

Work continued on wiki, human practices document, and refining protocols. Work continued on presentation.

Mammalian cell culturing and viability testing continues. Begin testing of transfection efficiency using EGFP coupled to the CMV promoter. We repeated the entire workflow for constructs which failed previous colony PCR screening. We then tested our new constructs using colony PCR. Colony PCR verified constructs were prepared for sanger sequencing. Constructs which passed sanger sequencing were grown up and freezer stocks were prepared.

By the end of the week, sanger sequence verified constructs were made for MT2_1, FGF-21, GADD45a, MT2_2.

Attempted to amplify the constitutive CAG promoter out of a plasmid obtained from addgene. Amplification failed for unspecified reasons, most likely mistakes in primer design.

Work continued on wiki, human practices document, and refining protocols. Work continued on presentation.

Mammalian cell culturing and viability testing continues. Continued testing of transfection efficiency using EGFP coupled to the CMV promoter. Began transfection using the sequence verified MT2_2 promoter EGFP construct.

We repeated the entire workflow for constructs which failed previous colony PCR screening. We then tested our new constructs using colony PCR. Colony PCR verified constructs were prepared for sanger sequencing. Constructs which passed sanger sequencing were grown up and freezer stocks were prepared.

By the end of the week, sanger sequence verified constructs were made for MT2_1, FGF-21, GADD45a, MT2_2, MT2_3, MT2_4, GADD153.

Work continued on wiki, human practices document, and refining protocols. Work continued on presentation.

Mammalian cell culturing and viability testing continues. Continued testing of transfection efficiency using EGFP coupled to the CMV promoter. Continued transfection using the sequence verified MT2_2 promoter EGFP construct. Began transfection using additional constructs and testing against chemicals of concern. Mass produced sanger sequence verified plasmids by growing up in bacteria and miniprepping, speedvac’ing, and nanodropping.

Work continued on wiki, human practices document, and refining protocols. Work continued on presentation.

Mammalian cell culturing and viability testing continues. Continued transfection using additional constructs and testing against chemicals of concern. Experimented with plate reader settings and media formulation. Mass produced sanger sequence verified plasmids by growing up in bacteria and miniprep, speedvac, and nanodrop.

Work continued on wiki, human practices document, and refining protocols. Work continued on presentation.

Mammalian cell culturing and viability testing continues. Continued transfection using additional constructs and testing against chemicals of concern. Experimented with plate reader settings and media formulation. Mass produced sanger sequence verified plasmids by growing up in bacteria and miniprepping, speedvac’ing, and nanodropping. Work began on biobricking. Selected a part from the distribution kit containing the pSB1C3 backbone, and transformed it into DH5a.

Work continued on wiki, human practices document, and refining protocols. Cleaned up the very large Project Design Notebook for legibility. Work continued on presentation.

Mammalian cell culturing and viability testing continues. Continued transfection using additional constructs and testing against chemicals of concern. Experimented with plate reader settings and media formulation. Experimented with use of fluorescent microscopy to measure expression of EGFP. Mass produced sanger sequence verified plasmids by growing up in bacteria and miniprepping, speedvac’ing, and nanodropping. Continued work on biobricking. Prepared the vector backbone by growing up in DH5a, miniprep, nanodrop, speedvac, nanodrop. Then restriction digest with XbaI and PstI. Restriction digest was followed by gel extraction. Inserts were prepared by PCR.

Work continued on wiki, human practices document, and refining protocols. We also mathematically modeled our data. Judging form was completed. Work continued on presentation.

Mammalian cell culturing and viability testing continues. Continued transfection using prepared constructs and testing against chemicals of concern. Experimented with plate reader settings and media formulation. Experimented with use of fluorescent microscopy to measure expression of EGFP. Completed work on biobricking. Prepared the vector backbone by growing up in DH5a, miniprep, nanodrop, speedvac, nanodrop. Then restriction digest with XbaI and PstI. Restriction digest was followed by gel extraction. Inserts were prepared by PCR. Inserts were inserted into the prepared vector backbone, ligated, and transformed into bacteria. Colonies were screened using colony PCR and then sanger sequencing. All constructs failed Sanger sequencing so entire workflow was repeated a second time. All constructs failed Sanger sequencing so entire workflow was repeated a third time. MT1 and MT2 (full) both passed sanger sequencing and were submitted to the biobrick registry.

Wiki was completed and all relevant documents were uploaded. Registry pages were completed. Work continued on presentation.

Completed data collection for the project.