On Monday we started our day greeting our buddy high schoolers that came from the MASI program at Purdue. We showed them through basic lab sterile technique and got their aid on our second, revised mini prep, which gave a high yield of our E.coli. Backbones. In addition, we finally managed to tie up loose ends with our gene problems and ordered all the parts we needed from the registry and IDTDNA.




On Tuesday we discussed our project progress with our PI, Jenna Rickus, and got her input on how to assay our enzymes that were transformed in our yeast. We then began running a growth curve test on our wild type yeast cells to set a standard to compare our modified yeast to. However, complications began to rise when the machine we used to count or cells experienced issues. Due to technical difficulties, we had to postpone this test for a later time. The high schoolers got the chance to learn more about lab technique when we decided to start our first E.coli transformations with our promoters, RBS, amp backbone, lysis agent, and terminator genes.




On Wednesday we went through a second round of E.coli. Backbone mini prep in order to stockpile on DNA for future experiments. However, due to restraints on mini prep supplies at the end of our second round, we resorted to creating our own buffers in the lab. In addition, only one of the transformations that we did yesterday went through successfully. Because of this, we were really bottlenecked for tasks and experiments we could do. Instead, we turned to coming up with possible themes for our lignin breakdown project, writing up more of our wiki information, and started on our yeast death curve.




On Thursday, we decided to attempt another E.coli backbone mini prep with our new buffers, but faced some lackluster results as the buffer we made was not optimal enough to extract the DNA properly. Also, the re-cultured E.coli. Were submersed in broth that didn’t have antibiotic resistance, so selective pressures reduced the amount of DNA that we could collect from a sample. Since we had to wait for our transformed cells to grow in their plates, the only lab related activity we had left was to create more competent cells for future experiments. The MTT assay kit we had bought also had very vague and confusing instructions, so we needed to refer to our graduate assistants and advisors for help and alternatives for the assay.




The majority of the work day involved running additional transformations with our yeast parts and mini prepping the promoter plasmids from E.coli liquid cultures. However, due to selective pressures and inefficient buffers, the mini prepping needed to be put on hold until more company buffers could be ordered. The rest of the time was spent working on the wiki, organizing our timeline, and researching future assays.