Review our Labwork.
Review our Labwork.
On May 30th we first received access to our lab along with a lab orientation from Dr. Deyholos. Throughout the next couple of days we began to organize the lab and took stock of lab supplies which had been both donated to us and left over from the previous year.
In the weeks following our wet lab members underwent lab training as taught by Gustavo and Ryan, two iGEMers from last year’s team who are experienced in molecular biology.
Simultaneously, our wet lab team was hard at work finalizing our constructs and preparing our protocols to start work in lab. Unfortunately, as a result of shipping times we were not able to start building our constructs until July.
We received a plate of B. Subtilis from Rosemary. This allowed for Danika and Mary to inoculate several liquid cultures of B. subtilis and prepare for when the receival of our DNA and primers the following week.
During this week several of our wet lab members attempted to transform B. subtilis via electroporation. Electroporation was attempted twice with no results before we decided to divert our focus and tried to electroporate E. coli instead.
Mary and Diego attempted to transform fuGFP into E. coli with electrocompetent cells which had been made a day prior, yielding no results. Believing that electroporation failed due to the use of old cells, Annika and Danika then attempted to transform E. coli with newly made electrocompetent cells however none grew.
After we received our raw sequences, Gustavo and Diego conducted a golden gate assembly to assemble our level -1 parts into level 0 parts. We were not able to try and confirm our level 0’s until the following week.
Diego and Mary attempted to electroporate E. coli with fuGFP and PUC19 one final time, also yielding no results.
Meanwhile, Gustavo and Diego used heat shock to transform all of our level 0’s into E. coli and then plated them for blue-white screening. However, blue-white screening failed due to use of X-Gluc during plating instead of X-Gal. In order to confirm the level 0’s, Diego and Gustavo instead ran a colony PCR of the transformed colonies overnight and inoculated several liquid cultures with the transformed level 0’s. A gel gave varied results, showing that the level 0’s for MWP, His 6-8, VP3, and CotB were not successfully transformed into E. coli and needed to be re-done.
Additionally, Annika and Danika ran a PCR to add flanks to our newly received raw VP3 and CotB sequences. They then proceeded with a golden gate assembly to assemble level -1 VP3 and CotB into level 0’s, however a gel showed that the original PCR had failed. Trevor and Mary re-did the PCR the next day and were able to confirm the flanks had been added with a gel, confirming we had successfully made our level -1 VP3 and CotB.
We received X-Gal at the start of the week, allowing Mary, Danika, and Gustavo to re-transform and plate MWP, His 6-8, VP3, and CotB for blue-white screening. Gustavo and Diego also made blue-white screening plates for the inoculated level 0’s. From their plates, Danika re-inoculated all of our level 0’s and re-plated R-linker 7-8, and terminator.
Colonies which grew on both plates and in liquid media (ThCl, PhCl, R-linker 6-8, VP3, and CotB) were miniprepped by Sammy and Danika. Colonies which either showed suspicious growth or no growth (MWP, His 8-9, terminator, His 6-7, and R-linker 6-8) were re-transformed by Mary and Danika and plated for blue-white screening.
Danika ran a colony PCR of the re-transformed level 0’s, however when visualized on a gel the bands were too faint to confirm any of the constructs.
Mary and Gustavo re-did the gel of the re-transformed level 0’s, confirming our level 0 VP3 construct and our level 0 backbone were successfully transformed.
Gustavo and Diego then re-did their golden gate assembly and re-assembled all of our level -1’s into level 0’s, excluding CotB and VP3. Afterwards, they transformed the level 0’s into E. coli via heat shock and plated them on blue-white screening plates. Blue-white screening was successful for all plates and Gustavo inoculated the white colonies in liquid media. Gustavo used the inoculated constructs to plate them on blue-white screening plates, however they overgrew and we were unable to determine if blue-white screening was successful.
This led to Danika taking the OD measurements of the inoculated level 0 constructs. Based off of the OD measurements, Diego then performed a 4-fold serial dilution of the constructs and plated them on blue-white screening plates. Once they had grown, Danika performed a colony PCR of pAGM9121, VP3, R-linker 7-8, His 6-8, His 6-7, and terminator. Mary then visualized the results of the colony PCR on a gel and we were able to confirm our level 0 VP3, R-linker 7-8, His 6-8, His 6-7, and terminator from the colony PCR.
During the week Gustavo and Diego also attempted to make our fuGFP BioBrick moclo compatible via golden gate assembly. They then transformed these into E. coli and plated them.
Diego and Annika ran a colony PCR of the remaining level 0 constructs (MWP, ThCl, and PhCl) alongside the other level 0’s and were able to confirm all remaining level 0’s by visualizing them on a gel.
Diego, Gustavo, and Annika performed a colony PCR of the E. coli containing fuGFP. However, upon running a gel, the transformation was found to be unsuccessful. Mary and Danika attempted to re-transform E. coli with fuGFP and plated a series of serial dilutions on blue-white screening plates, using up the rest of the fuGFP in the process. From these plates Gustavo and Diego re-did their colony PCR. Unfortunately, the colony PCR was unsuccessful and we were required to order the DNA sequence for a new fluorescent protein (hfYFP).
Diego and Danika then attempted to perform a golden gate assembly to assemble our level 0 constructs into level 1’s, however after running the reaction through the thermocycler and transforming E. coli with the plasmids it was realized that they had forgotten to include MWP and terminator in their master mixes.
Afterwards, Mary re-did the golden gate assembly of the level 1’s and transformed them into E. coli with Gustavo. Diego prepared a 9-fold serial dilution of the transformed cells and plated them. After the plates had grown, Mary and Danika performed a colony PCR of the level 1’s which Diego ran a gel of. Due to the annealing temperature being too high during the PCR reaction however, no bands appeared on the gel when Diego ran it. This process was repeated one more time, using level 1’s miniprepped by Diego and Danika. Again, the visualized gel showed no bands.
While Mary, Danika, and Diego were busy troubleshooting the level 1’s, Sammy began her work with B. subtilis. Sammy was successfully able to inoculate a liquid culture of B. subtilis, inoculate a liquid culture of E. coli containing pSEVA, and obtain the pSEVA plasmid from a miniprep. Sammy also attempted to transform B. subtilis but was unsuccessful. This was the first of many attempts to transform B. subtilis.
Sammy attempted to transform B. subtilis again but was unsuccessful and began to try and troubleshoot some of the steps of the B. subtilis transformations.
Danika and Mary also performed a troubleshooting PCR of our level 1 backbone pICH7732 and R-linker 7-8 which were then confirmed in a gel. Following this Danika attempted to dilute our level 1’s and ran a PCR reaction with the dilute samples. When visualized with a gel, all bands that appeared were in the incorrect places.
It was during this week that the UBCO campus was evacuated due to the McDougall Creek wildfire, rendering us unable to return to the lab until further notice.
For most of this week the UBCO campus remained evacuated, however we were allowed back into lab on August 25th.
Danika re-attempted a golden gate assembly of our level 1’s and transformed them into E. coli using heat shock. The transformed cells were plated and the following day Gustavo ran a colony PCR. A gel revealed that the golden gate assembly of our level 1’s were unsuccessful.
Danika ran a golden gate assembly to assemble hfYFP into a level 0 which Gustavo was then able to confirm with a PCR. This allowed Danika to transform the level 0 hfYFP into E. coli.
Danika also attempted to transform B. subtilis but was unsuccessful. Following this she began to try and optimize the B. subtilis transformation.
UBC Okanagan was one of many location impacted by the devestating fires that haunted Kelowna this summer. All members of our team evacuated the area and for the duration of the evacuation order.
Danika and Gustavo attempted to clone our transcriptional units into our pSEVA backbone however they were unsuccessful. Danika re-attempted to clone our TU’s into pSEVA and once transformed into E. coli, colonies were able to grow on blue-white screening plates. Unfortunately, when she conducted a colony PCR of the colonies none of the correct bands appeared on the gel.
Danika attempted to transform B. subtilis with her optimized protocol once this week and Gustavo also attempted to transform B. subtilis in chocolate milk, however neither transformation worked.
Gustavo cloned our TU’s into pSEVA with doubled concentrations of DNA. Danika attempted to run two separate colony PCRs of the TU’s in pSEVA however both gels showed incorrect bands, indicating that the transcriptional units had been ligated together incorrectly.
Danika cloned our transcriptional units into pSEVA and transformed them into E. coli. She then ran a colony PCR of the transformed E. coli and was able to confirm that two of our level 1 constructs, VHTC and VHPC, had successfully been cloned into pSEVA!
At the same time, Gustavo ran a golden gate assembly of our newly ordered level -1 CotB and hfYFP to assemble them into level 0’s.
Gustavo was able to verify our new level 0 CotB and hfYFP with a colony PCR with a gel.
Danika ran a golden gate assembly to construct YRC and VYRC and then attempted to clone them into our level 1 backbone. A colony PCR of E. coli transformed with these constructs showed that VYRC had faint bands at the correct positions, possibly confirming VYRC in our level 1 backbone. Danika then re-did the golden gate assembly and attempted to clone them into our pSEVA backbone, however a colony PCR showed that this was unsuccessful.
During this week we received bees from Armstrong Apiaries. This allowed Robin to start preparing for dissection and imaging. Robin also made a box for transferring the bees from their original box and into the bee box. However, upon opening the original box it was to our surprise that the bees which had been given to us were unknowingly infested with varroa. Upon the brood comb hatching varroa were released into the box and the health of many of the bees stagnated, leading to mass bee death. In fact, we found multiple bees infected with DWV as well.
Robin began dissections with some of the bees infected with DWV and fixed their tissues overnight. She then washed their tissues and embedded them in agarose, however she was unable to continue due to Dr. Rheault’s vibrotome being broken.
We received more bees, this time from Wild Antho, and Robin transferred the bees into a larger bee box.
Danika also attempted to transform E. coli with our pSEVA backbone, VHTC, and VHPC, however they were unable to be transformed due to our transformed plasmids being low in concentration.
Mary and Gustavo miniprepped our level 0 hfYFP and CotB for use in constructing our level 1 plasmids.