gBlocks containing Gal1-BS3 and Gal1-AmilC were obtained and resuspended in water.
gBlocks containing Scfv1 and Scfv2 (not used in final project design) were received and resuspended.
LB and gentamycin plates were made. LB broth was made.
Competent Dh5a cells were created and tested using pdonor 207 stock plasmid.
The pdonor 207 plasmid was miniprepped.
PGWB1 and PGWB2 vectors were miniprepped.
Gateway cloning of all constructs into pdonor 207 was attempted using the combined BP/LR reaction. No colonies were observed.
May 28th-June 1st
Gblocks were all gateway cloned (BP) into the pdonor 207 plasmids. Colonies were yielded on each plate. Primers designed for validation of the parts in pdonor 207.
June 4th-June 8th
The pdonor plasmids were sent off for sequencing and sequences were confirmed.
Gateway cloning LR reactions were performed using the PGWB1 and PGWB2 plasmids, along with AmilC, BS3, Scfv1, and Scfv2.
June 11th-June 15th
Colonies mini-prepped using the GENEjet mini prep kit.
PGWB1 and PGWB2 plasmids were screened using Vspi, incorrect except for Gal1 BS3 PGWB1.
Began planning first experiments of agrobacterium into Nicotiana benthamiana—because our system would be using toxin, we would first need a proof of concept showing we can transfer genes into plants.
Sponsoring PI reached out to Jennifer Brophy, who provided minimal CaMV 35S promoter with 6X UAS and VP-16 Gal4 activator plasmids in agrobacterium-compatible plasmids.
June 18th-June 22nd
Attempted to design plasmid encoding 6X UAS CaMV 35S promoter, but complexities were caused by repeated sequences that prevented gBlock synthesis
BS3 and AmilC cloning attempts into PGWB2 were conducted, no colonies again.
Gateway compatible multiple cloning site (7X RE) was designed and ordered as a gBlock.
June 25th-June 29:
Gateway compatible 7X RE was cloned into pDonor 207 plasmid using BP clonase.
Clones were validated using Kpni and Bglii performed, 7X RE was successfully cloned.
Primers designed to add 5’ site of Kpni to the CaMV 35S 6XUAS promoter and Asci to the 3’ end, as well as Xbai and Spei sites.. gBlocks for BS3 and AmilC created with Asci at the 5’ end and Spei at the 3’ end.
July 2nd-July 6th
The 6X UAS sequences were PCR amplified using Xbai and Spei forward and reverse primers, as well as kpni and asci 5’ addition and 3’ addition primers, and purified through a Genejet column.
The amplified fragments were digested using the handles for each primer that were added. The 7X RE plasmid was also digested with the same sets of restriction enzymes.
The fragments containing the amplified 6X UAS promoter sequences were gel purified and the DNA was extracted using the Genejet purification kit.
The promoters were ligated and transformed into 7X RE plasmid. A negative control was included.
Colonies observed on all plates.
July 16th-July 20th
The sequences plasmids were digested with Vspi to screen. They all returned as backbone.
We performed the interlab study. We helped Lambert to perform the interlab following our experience with it.
July 23rd-August 3rd
Cloning steps from July 2nd to July 13th were performed again, and yielded the 6X UAS promoter 7XRE plasmid.
In the interests of time, we decided to begin attempting to clone using the 6X UAS – CaMV 35S –GFP construct, pJAB 1504, and the VP-16 plasmid, pJAB 1489. BS3 PGWB1 was also transformed. First Agrobacterium cloning method only yielded pJAB 1489 colonies on the Rifampicin-Kan plates. Briefly, the transformations were incubated with DNA for 10 minutes, followed by flash freezing in liquid nitrogen. A 4 minute recovery in 1 mL of LB at 37 degrees was carried out followed by 4 hours of shaking at 28 degrees. Cultures were plated on Rifampicin and Kanamycin plates for 2-3 days before checking for colonies.
Agrobacterium transformations from August 6th-10th were repeated, but again did not yield colonies. A longer, 5 minute flash freeze was carried out, and BS3 yielded colonies.
From August 13th to 17th, we got down to our last stock of EHA105, so for this week we prepared competent cells for EHA105.
Interlab CFU was repeated.
Again, The experiment from the 13th-17th was performed, and finally the pJAB1504 plasmid was cloned.
For the 6XUAS promoter and BS3 resistance gene plasmids, they were cloned into the shipping backbone using Xbai and Spei sites.
Transient Expression of pJAB1504, pJAB 1489, and BS3 experiments were performed in Nicotiana benthamiana. Fluorescence and leaf death pictures were taken.
Helped Lamber test electroporator as part of collaborations.
Plate reader measurements taken for Lambert collaboration and Wiki design. Parts submitted.