Abbie, Ben Hardy and Alistair did another PCR on the CaMV promoter due to it being unsuccessful the previous day and then ran another gel. This time the results of the gel looked much more promising. After this initial step Abbie, Ben Hardy and Alistair performed another double restriction digest on the promoter and chloramphenicol plasmid. This was then followed by a DNA ligation. Abbie, Ben Hardy and Alistair also did a transformation with the wil type luciferase Biobrick. Ben Jevans and Gurdeep on this day went on to perform a miniprep of their cultures containing the site-directed mutagenesis product of the Ble gene. The isolated product was then ran on the gel which showed differing results depending on the medium the plasmid was extracted from. A nanodrop was done to check the DNA concentration of both samples that were tested. Ben and Gurdeep then did a PCR on the Ble gene from two different mediums; after this they ran the product of the PCR on a gel and unfortunately questionable results were obtained. Mario and Mark conducted a census of all the Biobricks that had been used and where they had been placed so lab work was kept in order.
Abbie, Ben Hardy and Alistair checked their plates which showed that he transformation they had done the previous day had been successful. They then went on to do a restriction double digest to isolate the luciferase Biobrick gene from the plasmid and then conducted a PCR on it. Next Abbie, Ben Hardy and Alistair did another transformation in E.coli with the luciferin regenerating enzyme and left it to incubate to be ready for the next day. Abbie, Ben Hardy and Alistair did another double digest on the CaMV promoter and pSBIC3 plasmid; this was followed by a ligation and then ran on a gel. Ben Jevans and Gurdeep re-did another PCR of their Ble gene mutants grown in the same medium used before. They then ran this on a gel which got some results. Ben Jevans and Gurdeep then grew three different site-directed mutagensis cultures from three separate colonies in LB broth overnight. Mario and Mark ordered some essential consumables including phleomycin antibiotic and dNTP for use in PCR.
Ben Jevans and Gurdeep gathered their cultures made from the previous day and did another miniprep. After this they ran there results on a gel and performed a nanodrop; results did not look to promising. Abbie, Ben Hardy and Alistair ran a gel on all their digested DNA parts; results yet again did not look to promising. They then did a nanodrop on the luciferase PCR product and pSBIC3 to determine the DNA concentration for future digests. Mario and Mark started researching ingredients for an assay buffer to test bioluminescence in the E.coli transformants. Mario and Mark went on to do some wiki work and help Abbie,Ben Hardy and Alistair with the moss transformation.
This morning Gurdeep worked on the wiki by adding additional context to the methods and lab journal. Gurdeep and Abbie then carried out a miniprep and gel electrophoresis on new cultures of the bleomycin gene, which unfortunately once again showed no bands, leading to no results. Ben Jevans made 15 new cultures from the colonies created from the Bleomycin resistant plates, ready for tomorrows miniprep procedure. Alistair also made new cultures from the transformed E.coli with GFP and YFP. Kimberley confirmed the images and lesson plan she would be using for her SAW project.