This year we planned to create 5 distinct BioBricks, due to multiple problems occurring in and out of the lab that will be discussed later, we were not able to successfully ligate any synthesised DNA into a plasmid for transformation.
SSPA
*Stringent starvation protein A* (sspA) is a protein native to Escherichia coli, it is normally expressed in the change from growth phase to stationary phase on the normal growth curve. It forms a homodimer to bind to genomic DNA, and acts as a transcriptional activator.- It’s sequence can be found on this Benchling link
CALCA promoter
This part was to show that our system could target and amplify human genes. Natively it initiates transcription of calcitonin protein A.- It’s sequence can be found on this Benchling link
gRNAs
The guide RNA (gRNA) is used to target the Cas9 protein to the calcitonin promoter. It was designed with 20 base pairs of complementary sequence, along with the scaffold sequence. In order to design the gRNA, we ran the CRISPR analysis tool from Benchling, and by following their tutorials we managed to design two gRNAs.dCas9
dCas9 stands for dead Cas9, as this version of Cas9 has a mutation that causes it to not have any catalytic action. This means that the Cas9 binds to the DNA through the help of gRNAs, and then just sits on the DNA, instead of cleaving it. We used the sequence from the Streptococcus pyogenes bacterium. We then had to break the sequence up into chunks as IDT only provided 1kb chunks of sequencing, so we decided to perform a gibson assembly upon the chunks to stitch the part together.- It’s sequence can be found on this Benchling link
dCas9 - sspA fusion
This is the same as the dCas9, however we have added a 10 amino acid linker and the sspA sequence to the end, meaning that when they are expressed, the two proteins should be made and folded attached to each other, so that both can perform their native functions together.- It’s sequence can be found on this Benchling link
