S-STEM Scholar Leilani Boren's Blog

 Introduction: This week there were multiple attempts to make progress with the recently assembled three way fragment that had been the focus of this semester. In an effort to optimize the three way assembly process, we in fact had trouble actually replicating the results from the previous week and led to several PCR reactions as well as a heat gradient that were all unsuccessful. Seeing as the proposed three way assembled fragment could not be gel excised then we are again at a standstill for the total assembly process. The 7kb plasmid from Deinococcus aquaticus was isolated again in an effort to conduct a single digest experiment to validate the identity of the 7kb fragment of DNA yet again, and provide samples for Jonathan to amplify a roughly 1100 bp section of the plasmid. A transformed D. radiodurans was also put through the plasmid isolation protocol to isolate a 6800 bp plasmid, so that the ampicillin resistance gene it included could be amplified as well to validate its i...

 Introduction:  This week involved two major projects, one being the obvious continuation of three way assembly of three fragments, left, tetr/a, and right, for eventual transformation of Deinococcus radiodruans. As a brief reminder, this eventual overlap product will be used to hopefully knockout the LuxS gene within the chromosomal DNA. This proof of concept would be ideal for future experimental knockout in D. rad . So far, we had many issues with the right fragment correctly overlapping both with tetr/a itself and the combination fragment left and tetr/a. We would frequently see overlap product on gels that seemed to be stuck in the wells, possibly forming a secondary structure. This week we conducted a heat gradient of right and tetr/a, to isolate this combination and attempt three way assembly by adding the left fragment, which has been much more successful this entire semester.  The other project pursued this week revolved around the mysterious 7 kb plasmid seen fr...