|During this period, we had a number of discussions on different possible topics. Suggestions included engineering microglia to target and kill brain cancers, targeting HIV surface proteins to bring about their destruction, or modifying microglia to target and engulf prions. Other ideas involved utilizing light receptors for specific purposes, or targeting biofilm formation. However, in the end we decided to focus on the circadian rhythm, with an aim to replicate this system in a mammalian system that could be used in iGEM. After some time developing a plan for this system, we discovered that the parts we required were covered by patents and so we would be unable to send anything we made in to the registry. As a result, we decided to switch to a bacterial system, and source some plasmids from Dr. Alex Ninfa (Michigan University) and Dr. Susan Rowland (University of Queensland).|
Clone Pick-up: We prepared 3ml cultures with ampicillin (100ug/ml) and chloramphenicol (20uq/ml). Colonies transformed with the plasmids pBAD33 and pET16b were picked and placed in the amp and chlor cultures respectively. They were then kept on a shaker overnight at 37C.
Glycerol stocks: for pET and pBAD33, 1.75mL of each culture were added to 0.5ml of LB+50% glycerol.
Minipreps of the pBAD33 and pET16b plasmids were conducted and gels were run to confirm their size.
PCRs were performed on pBAD, araC and lacI to add biobrick sites
3ml cultures of the E coli containing plasmids for GlnAp2, GlnG and the strain 3.3L*G (has lacI/glnG knockout) were prepared. Plates with colonies were obtained from Dr Alex Ninfa (Michigan University).
Glycerol stocks of plasmids from Dr Ninfa were prepared.
Minipreps of the plasmids containing GlnAp2 and glnG were prepared before we did a PCR on glnG to add BioBrick sites
Gel and purification of araC, lacI, pBAD and glnG. Nanodrop results:
araC = 195 ng/ul
pBAD = 152 ng/ul
glnG = 166.9 ng/ul
Digestion of araC, pBAD and glnG with EcoRI and PstI.