Experiments & Protocols
Each plasmid consisted of the R0011 pLac promoter, B0034 ribosome binding site and the programmable cell death part on pSB1C3 Each were verified by Sanger sequencing. E Lysis Gene (BBa_K1835500), Protegrin (BBa_K638000) and Tachyplesin (BBa_K1835501).Methods
Three biological samples of each sample was prepared. Transformed cells were grown overnight in antibiotic media. The cells were then diluted to an optical density of approximately 0.2. Experimental and control cultures were prepared. The colonies were allowed to grow for 15 minute then 1mM IPTG was established in the experimental colonies. Initial samples were prepared for spectrophotometry and returned to shake at 37 C. Further samples were collected at 1, 2 and 4 hours. Data points were given using a spectrophotometer, at OD600.
BsaI is a restriction enzyme with unique type 2 properties. What this means is that a sequence of DNA can be designed to have a desired overhang on each side. The BsaI will cut itself out of the sequence, as shown in the picture below:
After cutting its recognition sequence out and leaving the desired overhang, a gRNA with matching sticky ends can be inserted into the space. In other words, the gRNA will completely take the place of the BsaI restriction site.
The BbsI Restriction site can be added via PCR in order to create custom matching sticky ends to string multiple gRNAs in a row. This is known as Golden Gate Assembly.
BbsI is then used to snip out the gRNA to prepare for combining with more gRNAs in Golden Gate Assembly.