Week 13

9/15/13
After the Tecan run with various concentrations of theophylline to check how much can be added before becoming toxic to the cells, we decided to do another run with the pBAD+RS1+GFP and pBAD+RS2+GFP constructs with varying arabinose, theophylline and newly made M9 minimal media. Another run was set up later at night to test more of our cotransformants, pTet+TBS2+GFP/pBAD+RS1+TAL8 and pTet+TBS2+GFP/pBAD+RS2+TAL1. We are finally getting pretty much all of our parts together and in the backbone needed for submission to the registry. Unfortunately we cannot get them sequenced over the weekend, but they will get sequenced in the coming week.

9/16/13
Today was parts submission day for us. Much of our time was spent getting all of our parts pages together and getting our samples ready. Unfortunately, we ran into some issues with one of our riboswitches that had a PstI restriction enzyme site in it, making it not RFC 10 compatible. The site happens to be vital to the function of the riboswitch, so we decided to try sending it in anyways and seeing if an exception can be made. However, some of our constructs are likely incorrect because we used Standard Assembly to put them together, so more work may still need to be done.

9/17/13
Construction of our constructs still continues especially since some of the sequencing results that we got back today were less than desirable. In light of the PstI site in one of our riboswitch sequences, we are using SOEing when doing construction with that part instead of Standard Assembly. Site directed mutagenesis of our pTet+TBS1+GFP is being performed again, since the sequencing came back incorrect. We had another meeting with our advisors where were made a new outline for our presentation, which we felt was slowly losing focus. We also discussed the recent data that we have been getting from our various Tecan runs over the last week or so. We decided that we should focus on characterizing our working constructs with more combinations of our inducers than we have been doing thus far.

9/18/13
According to the recent Tecan runs, arabinose and theophylline seem to be the only significant effectors on our construct. Our sequencing of several of our assembled constructs was a little perplexing, so we will continue assembly. However, next week we will spend more time working on the wiki, presentation and poster as the Jamboree approaches. We will also continue to do characterization runs of our assembled constructs.

9/19/13
We got some more sequencing back today, so most of the parts that we sent in to the registry are now sequence confirmed. Some of the constructs that we thought were incorrectly assembled due to the PstI site in one of our riboswitches turned out to have some correct colonies as well.

9/20/13
We tried to improve upon the confusing results of our Tecan run by trying many different conditions, along with fresh cultures of our cotransformations. We knew that pTet+TBS+GFP was functioning correctly, but our riboswitches and arabinose promoter were not functioning as expected. To deal with this, we set up a plate with different concentrations of high and low amounts of theophylline and arabinose. We kept the amount of aTc in each well at 100 ng/ml. We also used different types of media such as LB, TB, and M9. Finally, we also checked to see if our pBAD+RS 1+GFP and pBAD+RS 2+GFP constructs were functioning properly.

9/21/13
After looking at the run from last night, both the pTet+TBS 2+GFP+pBAD+RS1/2+TAL8 cotransformations appear to show the response to arabinose and theophylline that we were expecting. However, the pTet+TBS 1+GFP+pBAD+RS1/2+TAL1 cotransformations did not show any significant fluorescence, so it appears that the error in our oligo did prevent the functioning of pTet. Based on the sequencing of pTet+TBS 1+GFP, the SDM finally worked. In other words, the error produced by oligo synthesis has been fixed. We most likely have a functional Tet operator now. We will now use this plasmid in our cotransformations for characterization. We also checked the sequencing of TAL 8 to see if there was an issue of mislabeling, and there was not. In light of the promising data from our broad screening run and confirmation of our plasmids, we set up a more indepth run for pTet+TBS 2+GFP+pBAD+RS1+TAL8 with more combinations of our 3 inducers: 6 different arabinose concentrations, 5 theophylline and 3 aTc.