Team:Queens Canada/Notebook/Week5



Notebook - Week 5

DateProtocolPeopleDNA (if relevant)Quantities and Parameters (if relevant)Notes on Protocol/ Gel Lanes
Week 5     
May-28DNA RehydrationVictor, Faisal, David, PhillipBBa_K346001, BBa_K190019, BBa_K346003Two trials for each Biobrick 
May-29DNA RehydrationVictor, Hope, Maggie, BeiniBba_346007, BBa_1721002Two trials for antigen 49, two trials for lead binding protein 
May-30Heat Shock TransformationVictor, Dan, FaisalBBa_k346007, BBa_K346001MerR, Antigen 43 
 MiniprepVictor, Dan, Faisal MerR, Antigen 43 
May-31DNA RehydrationVictor, Hope, BeiniBBa_k346003, BBa_E0040, BBa_J52008  
 Liquid Culture    
Jun-01MiniprepVictor, Tim, PhillipBBa_K746007, BBa_K23000, BBa_K123001, BBa_J52008, BBa_E0045BisdA, Antigen 43, GFP, Luciferase

Monday May 28

Throughout the day, we mostly worked on sponsorship, but we also spent some time in the lab performing plasmid dehydration and growing colonies on plates for three different BioBrick parts. Essentially, the next few days in the lab are going to consist of isolating plasmids from different BioBrick parts. The parts we incubated today are BBa_K190019 (fmT), BBa_K346003 (RBS(B0032)+MBP) and BBa_K346001 (MerR). On the wiki side of things, Phillip edited the wiki so that the Notebook section is nearly ready to go up!

Tuesday May 29

In the morning, we used GENtle 2 to analyze the D1 and D2 domains for the fliC gene in order to determine the primer sequence for digestion and PCR. We also spent some time today working on DNA primers in preparation for the PCR of the BioBricks that we plan to order. Beini, Maggie, and Victor spent time in the lab today working on miniprep for BBa_K190019 (fmT), as it was the only part that managed to result in colonies. Neither of the other two parts resulted in colonies. We also did dehydration and heat shock transformation for two new parts, Antigen 43 (BBa_K346007) and lead binding protein (BBa_I721002), which are grown with chloramphenicol and ampicillin resistance respectively. We also finally set on a (tentative) name for our project: ChimeriQ. Some of the other ideas were E.coFliC, sQaffolD3, BioremeD3ation, and fli.meriC.

Wednesday May 30

Today, we spent some time checking over our DNA primers for PCR. In the afternoon, we had a meeting with our faculty advisors, Dr. Chin-Sang and Dr. Bendena. We discussed the current progress of our project and discussed some of the problems we have had with our lab procedures. Victor, Faisal, and Daniel worked in the lab today and performed miniprep with fmT colonies. They retried rehydration protocols for Antigen 43 and MerR BioBricks using different protocols to find out which protocol is most successful. They also tried to centrifuge liquid cultures and use the pellets for better transformation.

Thursday May 31

Today, we spent time finalizing our DNA primers for PCR. We worked on adding and checking our linker sequences that we added to the primers in order to have the parts that we characterize work properly with the C1 and C2 domains. We also spent some time checking over the linker sequences in the C1 and C2 domains to make sure that everything worked correctly. In the afternoon, we looked at the results of the rehydration protocols for Antigen 43 and MerR BioBricks. It turned out that the Antigen 43 had colonies, but the MerR did not. We also performed rehydration of two more BioBrick parts and performed liquid colonies of Antigen 43.

Friday June 1

We worked on researching where to get 6xHis-tags, as well as nickel resins, for future purification of our proteins. We also read some interesting articles on flagellar nanotubes.