Team:Calgary/28 July 2010


Wednesday July 28, 2010


The progress on learning Autodesk Maya seems to be going quite smoothly. I spent much of the day reading through the basic tutorials. I now know how to manipulate and create various polygonal and smooth-surface shapes. Tomorrow I will continue learning the program in polygonal modelling.


Today, I started the constructions of degP and CpxR promoters (K135000 and K239000) with the RFP and GFP constructs (I13507 and I13504) using the restriction enzymes XbaI, SpeI, and PstI. The constructions were done into AK plasmid backbones. As well, I started the PCR with primers that were previously designed by Alex and Patrick to take the CpxP promoter from the E. coli genome. The PCR finished but it ended too late to run on a gel. Tomorrow, that gel will be run. I continued contacting companies about possible sponsorships for our project. Our current sponsors will be posted on the website soon.


This morning Raida and I ran gels of my three PCR reactions from yesterday. One of the reactions with the malE-BBK primers showed amplification of bands at approximately 1200 base pairs, where we woule have expected. The other reaction with this primers (at a higher concentration) showed no amplification. We tried doing a gel extraction of the one set of PCR products, however we combined the wrong lanes, so we abandoned this initiative. Our reaction with the malE signal sequence deletion showed amplifiaction of a band of approximately the right size, however only in the last lane (the highest annealing temperature in the gradient). There was also a fairly storong band int he negative control lane however. Because of this, we will try running the reaction again, at a higher annealing temperature to see if we can get amplification and a clean negative control lane. We also set up a PCR with these same primers at a lower annealing temperature, as this gave us results with similar primers when we were trying to BBK malE. Tomorrow we will go back to our PCR products from last week and try to get malE and malE31 into a TOPO blunt vevtor in order to facilitate the process of biobricking these suckers.

Today I also spent some time looking into what reagents/ equipment we want to ask for from Corning Life Sciences and from VWR. I figured out how to get a free trial version of Photoshop for use in the Wiki design. I also made overnight cultures from the plates of my I0500-I13507 and I0500-I13504 constructions in order to try inducing them with arabinose later in the week.


The plates with plasmid switch from July 27, 2010 grew. I did a PCR of 11 colonies of B1 and 11 colonies of C1. I ran a 1% agarose gel of the PCR products with the master mix as controls. The PCR unfortunately did not work. B1 DNA gave wrong sized bands but C1 gave me no bands at all. Seems like there was no replication for the C1 colonies. I will not provide pictures of gel because it has absolutely no bands. Therefore I will reconstruct tomorrow. I also ran a restriction digest of the different versions of I0500 (C1-D1, C1-D2, C2-D1, C2-D2) with EcoRI/PstI and SpeI/XbaI that we have sitting in our freezer to check which of the plasmids have the right size bands. The gel showed bands at about 1600 bp which is the incorrect size of bands. I also did research on what factors are responsible for inclusion body formation and how the mechanism of inclusion body formation differs in each case.