However most of the parts did not come with the repressor and therefore we only picked BBa_K1758333 (lead) and BBa_K1758343(mercury). We performed the following:

Extracted the plasmids from the toxins case with the corresponding metal promoter
Transformed the plasmids into 9 different competent cell cultures.
Resuspended the cells with LB and added SOC after the heat shock and plated them on chlor plates
We streaked plates for isolation for later use
We picked colonies off the plates and made liquid cultures
Made toxin dilutions and added cells with the metal promoter plasmids
Let the culture grow overnight
Set up a 96 well plate and added all the different components along with four blanks of LB

96 Well Plate Set-Up

In 5 mL culture:

Hg/Pb

Low: 0.5mL of 10 mg/mL stock = 1ug/uL
Medium: 0.5mL of 1 mg/mL stock = 0.1ug/uL
High: 0.5mL of 0.1mg/mL stock = 0.01 ug/uL

Cr

Low: 2.5uL of 0.1 mg/mL stock = 0.05ug/uL
Medium: 2.5uL of 1 mg/mL stock = 0.5ug/uL
High: 2.5uL of 10mg/mL stock = 5 ug/uL

Cu

Low: 1uL of 10 mg/mL stock = 2ug/uL
Medium: 2.5uL of 10 mg/mL stock = 5ug/uL
High: 5uL of 10mg/mL stock = 10 ug/uL

Some cells with other metal promoters were emitting fluorescence even before we induced the cells with the toxins. As a result, the metal promoters are unaffected by the inducers and do not work effectively.

Looking at the graph, with increasing concentration of relevant levels of inducer(toxin) the relative fluorescence units are not increasing as expected.

As the inducer level increases we expect induction to increase. However for BBa_K1758343, all the induced RFU measurements for low, medium, and high concentration were less than the uninduced RFU measurements. The fold change was relatively the same for all levels of inducer which contrasts the trend that is shown on the Bielefeld 2015 website. Therefore, we can conclude that the mercury promoter did not work.

Furthermore, in regards to BBa_K1758333, there is a fold change between the GFP uninduced lead and GFP low induced lead RFU average measurements. But after looking over the two replicate GFP low induced data measurements, one of the values is slightly larger than the other so the data values are inconsistent. Therefore, we cannot make any conclusions about the fold change in the GFP mercury low induced RFU measurement. The fold change values for the GFP medium and high induced RFU measurements showed that the fluorescence expression was decreasing as the inducer concentration increased. Therefore we can conclude that the Lead promoter did not work. Team Bielefeld also concluded that there were no significant differences in fluorescence response for the lead promoter.

Team:Florida/Contribution

Characterization or Contribution

96 Well Plate Set-Up

In 5 mL culture: