The first few steps of the project involved purifying and checking both of the genes for Cas9 and aTcdB. This involved purifying the products of PCR and running them on agarose gels, as well as using a spectrophotometer to identify the concentration and purity of DNA recovered.
1. pHis1522-aTcdB
| Sample |
Concentration (ng/μL) |
| A |
135.5 |
| B |
170.2 |
2. pHsp70-Cas9
| Sample |
Concentration (ng/μL) |
| A |
32.9 |
| B |
33.3 |
| C |
118.3 |
The presence of 4kb bands confirms the strong presence of Cas9 in at least some of the samples.
The genes were then ligated into vectors and transformed into JM109 E. Coli cells in order to replicate the vectors. The bacteria were left to grow overnight, and then the plasmid was isolated from them.
pHsp70-Cas9
| Sample |
Concentration (ng/μL) |
| A |
45.7 |
| B |
46.3 |
| C |
40.4 |
Finally, the NLS-tagged Cas9 PCR product was combined with the linearized pHis1522-aTcdB vector in a Gibson assembly reaction. This reaction was transformed into NEB 5-alpha E. coli. Several transformants were used to inoculate liquid cultures, whose plasmids were subsequently isolated.
Concentrations of pHis1522-Cas9-aTcdB Minipreps
| Sample |
Concentration (ng/μL) |
| 1 |
49.9 |
| 2 |
72.1 |
| 3 |
1702.6 |
| 4 |
426.6 |
| 5 |
42.7 |
A PCR to amplify the Cas9 portion was performed to confirm sucessful assemly. Below is the gel of samples 1-5. A faint band (not clearly visible in image) at ~4.2 kb in sample 3 indicates Cas9's presence.
In addition to the creation of the Cas9-aTcdB protein, the lab also worked on the creation of a template for the easy customization of the gRNA for T7 driven transcription. This construct was obtained as a gBlock, amplified, and inserted into the pSB1C3 vector with restriction cloning using EcoRI and PstI.
Colonies lacking RFP were used to inoculate liquid cultures, whose plasmids were ultimately purified and sequenced. Sequencing confirmed successful insertion. This construct now constitutes our Biobrick part BBa_K1818000.
Unfortunately, an attempt to insert a pair of phosphorylated annealed oligos into this template was unsuccessful, as confirmed by sequencing which shows that the BbsI did not cut the vector or that the vector re-ligated.
