Team:Northwestern/Collaborations
COLLABORATIONS
UNIVERSITY OF NEBRASKA-LINCOLN
We partnered with the University of Nebraska-Lincoln (UNL) to help characterize part BBa_K584000, a pBAD promoter upstream of the GFP gene. We transformed and tested this part with a procedure drafted by UNL. This part is inducible with L-arabinose, so our objective is to perform a plate reader assay to quantify fluorescence/OD with varying levels of L-arabinose added to the cell culture. Click hereto visit UNL's Wiki and to find out more about this procedure.
RESULTS
As illustrated in Figure 1, fluorescence standardized over OD600 increases steadily from hours 1 to 3, but experiences a drop during hour 4. At hour 5, the 0.2% of L-arabinose sample seemed to produce the most fluorescence from BBa_K584000. These results are consistent with a previous characterization by the K.U.Leuven 2011 iGEM team. K.U.Leuven found that adding 0.2% arabinose yielded higher amounts of fluorescence than 2% arabinose. Click here to read more about that characterization.
Figure 1: Data on induction by L-arabinose on BBa_K584000.
Improvements:
While following their protocol, we developed a few suggestions we believe can improve the procedure.
- Kinetic reading of OD and fluorescence
Figure 1: Data on induction by L-arabinose on BBa_K584000.
Doing a kinetic read of OD and fluorescence shows the continuous cell growth and increase in fluorescence, which can be helpful especially in shorter experiments where there are a smaller amount of reads. This helps the data be more detailed and can remove ambiguity, as seen in the hour 4 data point in Figure 1. Ultimately, a kinetic read allows for more time points closer together, which can help the data be clearer and better support the conclusion.
- Optimized cell ratio for transformation
Depending on the chassis used, the cell ratio used for transformation is important for effective transformation. We used a different chassis, and therefore had to adjust the cell ratio. Additionally, certain cells may be more or less suited to transformation.
TRANSFORMING mCHERRY
The University of Nebraska-Lincoln helped us troubleshoot transforming part BBa_J04454. UNL successfully transformed mCherry using 1 µL of DNA and 50 µL of Gene Hogg as a chassis. This step was crucial to completing our final design: using mCherry as an internal reference of cellular health in conjunction with GFP to reference DNA repair. More information about how we intended to incorporate this part can be found in our Future Applications sections of our Results page.
