On Monday, we spent the day researching alternative assays to perform, as well as investigating alternative killswitch pathways. We also inoculated the E. Coli test colonies in LB+Chloromphenocol broth to determine the success rate of our promoter plasmids. Before plating the liquid culture, we also wrote out and clarified many of the procedures we’ve performed and others yet to come.




On Tuesday (upon the arrival of more buffers), we miniprepped the killswitch’s RBS, Backbone, and Terminator DNA sequences, preparing them for assembly. Also, our practice yeast transformation was discovered to be a failure, as our yeast broth was contaminated through unknown means. Bottlenecked by the lack of IDT DNA, we decided to re-write the majority of our procedures in paragraph form, for easier addition to our wiki.




On Wednesday, we discovered yesterday’s miniprepping to be a failure, prompting a second effort, as well as miniprepping the promoters for eventual transformation. Also completed today was the first preparation of our switchgrass sample via milling. We began the transformation of our RBS+Terminator genes into competent E. Coli cells. Outside of the lab, we began brainstorming fitting titles for our project. Considering the Pirate theme, “Dead Lignin Tell No Tales” seems to be the current leader.




On Thursday, our G-Blocks arrived from IDT! We spent the afternoon diluting and re-suspending the DNA samples, and began PCR in the late afternoon. We also started our second attempt at the RBS+Terminator assembly (as Wednesday’s failed… the use of competent cells places our DNA under suspicion.). We also began electrophoretic gel construction, in order to more fully check our results and discover the source of the error.




On Friday, we underwent gel imaging training, in order to obtain clearer results from the gels (the ethidium bromide used in the gel fluoresces under UV light, allowing easy identification of DNA bands). Then, we ran gels for last night’s PCR products. We also poured more LB/LB+Chloromphenocol broth, for use when testing the transformations of E. Coli ligations (also performed today). We also discovered that the assembly of the RBS to the terminator was a failure, prompting the research and purchasing of new killswitch components. Also, due to our Graduate advisor running a workshop next week (rendering BIND134 unusable), we moved supplies downstairs for easy access.