Cas9 Project

Promoter Project

**The components of this project are the same as those in Cas9 and because of this, the notebook starts in August**

Aug 11-17

Re-isolated cytoskeletal promoter, Actin, from Arabidopsis thaliana genome. Actin was unsuccessful so it was PCR amplified again. A genomic isolation was performed from Arabidopsis leaves. Gel extractions of Myosin, Actin, MMV, and ClCuV.

Aug 18-24

PCR of myosin and profilin were both successful. CBAR 1 and CBAR 2 were performed again as described in cas9 notebook. In addition, FiMV promoter was put together again with CBAR. All were successful except the CBAR’s that were rescue PCR amplified. It was determined that the annealing temperatures were not compatible for CBAR 1 and 2 amplification so new primers were ordered. FiMV was successfully PCR amplified and was gel extracted. An intermediate CBAR of CMV35s(2) with mCherry (CBAR 3.5) was conducted and successfully amplified. Each component of the construct was CBARed again successfully and a CBAR of those parts was conducted to assemble the entire construct. It was unsuccessful.

Aug 25-31

More intermediate CBAR’s were conducted to piece together CBAR 1 with CBAR 2 from above, named CA. Also, CBAR 3.5 and Nos3 were joined together and named CB. Both attempts were unsuccessful and were tried again.

Sept 1-7

Began biobrick assembly by PCR amplifying Actin, Profilin, ClCuV, MMV, CesA 7, CesA 8, and Myosin with new primers that had the correct restriction enzymes on them. Continuation of trying for successful CBAR reactions with a series of new calculations, new primers, and new annealing temperatures during rescue PCR.

Sept 8-14

Restriction enzyme digest of all of the biobrick components and pSB1C3. The was some success with CBAR 1 and CBAR 3.5, but nothing could be amplified to standards.

Sept 15-21

Biobrick parts were individually ligated into vectors and transformed. Colonies were picked, cultures were grown and mini prepped to be sent to sequencing. Continued to troubleshoot CBAR reactions and look into a different method for assembly.

CesA Project

Butanol Project

Vanillin Project