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Team:UC Davis/Notebook/Week 11
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Mutant screening is going very well this week! We've gone from a 96 well screen of our R0010 mutants to having 7 reliable and ranged mutants. We have also started to move our characterization parts into plasmids with a terminator (pSB1C3). This will also be very useful when we ship parts to the registry.
The digestion (Promoter+E0240+I13453+B0034) from last Friday looked good on a gel. After gel purifying it, we were finally able to ligate in the repressor mutants.
Today we also made more LB+Carb plates.
The new Tecan schedule was put into place today. By inoculating liquid cultures in a 96-well plate twelve hours before a schedule Tecan run and then incubating at room temperature with shaking, one run can be prepared while another is being analyzed by the machine. To start this schedule, a 96-well plate with 87 R0010 mutants in our (promoter)-E0240-B0034-I13453-C0012 construct was inoculated in the late evening for a Tecan run on Tuesday morning, along with wild-type control (WT R0010-E0240-B0034-I13453-C0012), LB control, and DH5-a control.
The next goal in construction is to take our 9 screening parts and putting them in a new backbone. Our construct is designed to end with a repressor, where it should end with a terminator. It is currently in pSB1A2 which does not include a terminator. The plan is to cut all of our parts at EcoR1 and Pst1 and ligate them into the pSB1C3 (also cut at EcoR1 and Pst1). Once this is done, we can go even further with our characterization.
The 96-well plate for screening R0010 mutants was put into the Tecan for scanning early in the day and another plate of C0012 mutants was prepared. After around twelve hours, it was removed, and the data stored for analysis. We prepared scripts in Octave that would analyze data from 96-well mutant screening plates and deduce which liquid cultures contained DH5-a with mutant promoters outside the observed range for wild type.
This time, the mutants were real. As shown in the photo, many of the wells in our 96-well screening plate showed GFP activity that was far outside the range of the wild-type control. 29 promising mutants were selected from those which had activities different from wild type by at least 1.5 standard deviations (highlighted in green instead of blue on the graph), and another Tecan run was set up to analyze these in triplicate.
We were also able to select 29 C0012 mutants from our screen, despite the arabinose induction level on the plate being far too high. These were run in the Tecan overnight.
Last night, we stayed late after our 7 hour digestion to run the parts of a gel and purify them. However, it was 1:00 AM at this time so we decided to save the ligation and transformation for this morning. We transformed and plated all of our screening/characterizing constructs in pSB1C3 and will find out tomorrow if it was successful!
The next round of Tecan data showed that the 29 selected promoter mutants were well-varied in their GFP activities. This confirmed our screening process, solidifying our confidence in the mutants we selected. From the 29 now 'known-good' promoter mutants, we selected seven to run an extra set of screens on. We prepared four different reaction conditions under which to test these seven mutants (and wild-type R0010): maximal promotion of GFP (10mM IPTG), maximal repression of GFP (1% arabinose), medium promotion of GFP (no IPTG or arabinose) and medium repression of GFP (.3% arabinose).
C0012 data did not look as promising. No arabinose was added to the media, causing full repression of our C0012 repressor mutants and, therefore, nearly constitutive expression of GFP in almost all wells. The 29-mutant triplicate screen of C0012 was set up to be repeated with .1% arabinose induction.
-Friday 8/26/11--
R0010 data shows the expected range of GFP activity across multiple IPTG induction levels. Arabinose induction levels were too high to asses promoter function, but as expected GFP expression was very low under high repressor concentration conditions.
We realized that although our R0010 promoter mutants were good, but the screening plasmid had no terminator. To combat this, we began the process of cutting our constructed parts out of their carb-resistant backbone and placing them into psB1C3, a cholo-resistant backbone with a terminator included. Further screening of the R0010 mutants was put on hold until they could be inserted into the proper backbone and transformed into E. coli strain BW22826.
The 29 selected C0012 mutants were run in the Tecan overnight at the correct .01% arabinose induction level.
The 'known good' R0010 mutants were cut at E+P to be inserted into the psB1C3 backbone.
-Saturday 8/27/11--
The C0012 repressor data showed that our 29 selected repressors did not possess an ideal range of activity. Most had GFP activity much higher than wild-type -- as expected of random mutation -- but the spread of activity levels was too narrow to provide much functionality to a library. Because of this, a new C0012 84-mutant repressor screen was prepared and run at .01% arabinose induction, along with wild-type controls at 0% and .01% arabinose induction. This should give us a good idea of expected maxima and minima for GFP expression levels compared to wild-type C0012 repressor and, effectively, no repressor at all.
-Sunday 8/28/11--
C0012 84-mutant screening was run in the Tecan, once inoculated directly from replica plates, and once from the liquid cultures that were in the Tecan itself, which were stopped in the middle of exponential phase to serve as a liquid culture inoculum before being returned to the Tecan to complete the analysis cycle.
R0010 promoter mutants #1-7 (our 'known good' mutants) were gel-purified, ligated, and transformed into BW22826.
