Team:Calgary/9 August 2010


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Monday August 9, 2010

Chris's gel of CpxP and certain parts that were found in a box. Lanes 6-7 are of CpxP in pSB1AK3 and Lanes 8-11 are CpxP promoter in pSB1AC3.

Happy Birthday Jeremy!!


Today I did a construction of I0500-B0034 with MalE31 that was biobricked by Emily. I cut I0500-B0034 (pSB1AC3) with XbaI and PstI and I cut MalE31( psb1AK3) with SpeI and PstI. This disgest was followed by phosphatasing the vector and ligating the two parts with qucik ligase. I plated on AC to select for the cells which have the vectors.

I also read some more papers regarding the relationship between pH, temperature and inclusion body formation. I also went through some tutorials for MATLAB in order to help me define parameters and equation that will make the actual model.


Today I picked up the stuff I did over the weekend. Which included ligating, transforming and plated the bacteria for the pRFP plasmid switch of pSB1AK3 and pSB1AC3. I am also doing the restriction enzyme again this time with the right NEB buffer for the plasmid switch. This also included antarctic phosphatase and am leaving in the freezer until tomorrow. I also finalized the t-shirt print ready designs, all we have to do now is contact a t-shirt company. And briefly fixed several of the planned wiki page. Some of my time also went towards looking over our mascot costume that came in today.


Today I did a restriction digest of constructions (I0500+ I10504 and I0500+ I10507) done by Chris to verify that the construction worked. The gel can be seen to the side. As it can be seen, the construction of the I0500 with I0504 shows proper bands and the right lengths- one of the size of the vector, and the other of the constructed parts. However, the construction with I0507 did not show proper bands. To further verify the first two bands, I have also done a colony PCR with the BBK primers.


Today I ran a gel of my two gradient PCR's from last night, one of malESSdel and one of malE31SSdel. The gel of malESSdel looked really good, with very strong bands exactly where we had expected them (~1.2KB). The last lane, the negative control also looked good. There were some nonspecific products (very small fragment size), so we will PCR purify these and hopefully get rid of those. The gel of malE31SSdel however showed no amplification whatsoever, so we will have to restart that. I set up two ovenright gradient PCR's, one of malE31SSDel using a different colony than last time, and one of the same colony of malE31SSdel using varying concentrations of Mgcl2. I also made overnight cultures of all of my EcoRI/ PstI and XbaI/SpeI cut colonies of malE and malE31. I will miniprep these tomorrow morning.


Today, I set up a digest with XbaI and PstI of the CpxP promoter in the AK and AC plasmid that was inserted unpiurified. the gel of this can be seen to the right. The lack of any bands around the 250 bp region indicates that the cuts were unsuccessful. As well, I continued to contact companies about getting possible lab materials and funding for our team.


Looking into aggregate body formation, still. The search is turning somewhat fruitless. Also trying to determine which protein should be modelled.