Part:BBa_K911003:Experience

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Applications of BBa_K911003

This riboswitch came to us from the Yale team in the following construct:

The lysine promotor is constitutively active in both e.coli and bacillus, so expression of β-galactosidase should only be affected by the rate of transcriptional attenuation by the fluoride riboswitch.

To verify that this part worked before undertaking more complex assays, we decided to run an assay with X-Gal to give us some qualititative data about its function. The results of this assay are shown below:

The numbers on the tops of the tubes represent the concentration (in mM) of NaF in each culture. The amount of blue is proportional to the amount of β-galactosidase activity present in the culture, which in turn is regulated by the riboswitch. This qualatative assay demonstrated that our part worked.

The next step was a more quantitave assay using a 96 well plate reader. This was in line with the assays done in the original paper by [http://dx.doi.org/10.1126/science.1215063Breaker et al (2012)]. In the paper they carried out a Miller assay, but unfortunately we lacked the filters and so were only able to record the A450nm. We feel that this was close enough in wavelength to be a suitable proxy for the A420nm reading required to assay the o-nitrophenol produced. We ran an initial assay and indicative results are below. All the A450 bar charts are based on two repeats, error bars are not included as they would not be statistically significant.

The timecourse of the first run of the β-galactosidase assay using crcB knockout B. subtilis transformed with the fluoride riboswitch

A450nm readings after 420 minutes with crcB knockout B. subtilis transformed with the fluoride riboswitch

A450nm readings after 420 minutes with B. subtilis strain 168 transformed with the fluoride riboswitch

A450nm readings after 420 minutes with E. coli carrying a plasmid containing the fluoride riboswitch

This data seems to show a trend, that at low fluoride concentrations there is a positive relationship between concentration and A450, the A450 then plateaus, and then at high fluoride concentration the A450 drops again, this gives the overall data a bell shape. A possible explanation for this is that there are two conflicting effects, an increase in fluoride leads to an increase in β-galactosidase expression which, in turn, leads to an increased A450. However, the increased fluoride concentrations begin to become toxic and so limit the cell’s ability to produce β-galactosidase, leading to a reduction in A450. It would also seem that the linear region of the initial positive relationship seems to lie between 0μM and 100μM. We therefore repeated the assay, with a longer time course, and with greater resolution in this range, in order to see if the original trend could be reproduced, and also to better quantify this initial correlation. Included below are the results for the repeated assay.

The timecourse of the second run of the β-galactosidase assay using crcB knockout B. subtilis transformed with the fluoride riboswitch

A450nm readings after 790 minutes with crcB knockout B. subtilis transformed with the fluoride riboswitch

A450nm readings after 790 minutes with B. subtilis strain 168 transformed with the fluoride riboswitch

A450nm readings after 790 minutes with E. coli carrying a plasmid containing the fluoride riboswitch

For clarity, and to act as an internal control, the bar chart results are expressed as a percentage change from the same culture, after the same length of time, but with no fluoride added. This assumes that the cultures behaved the same across all fluoride concentrations over the assay, i.e. cell density and productivity were unaffected by the fluoride. This sort of assumption would no longer be needed if a ratiometric system were used, part of our motivation for developing ratiometrica. This data seems to confirm the initial findings, as well as giving greater resolution in the 0μM to 100μM range. The results for higher fluoride concentrations do not seem to quite fit the trend, but as this is consistent across all the cell types it is possible that this was due to errors in making up the solutions, rather than actually being representative of the riboswitch. This data is very encouraging as the WHO safe limit for fluoride is 1.5mg/L (according to [http://dx.doi.org/10.1016/j.envpol.2006.05.007Farooqi et al]) which works out at 79μM. This means our riboswitch is at its most accurate around the safe level for humans.

It should be noted that we expected a greater difference in sensitivity between the knockout Bacillus strain provided by Yale, the 168 strain we were using, and the E. coli we used. In the original paper there was a significant, nearly 100-fold, difference in sensitivity between the knock and wild type bacillus strains, but our data did not reproduce this. However, we feel it is actually very positive as it means our part is consistent across multiple chasses, and that it has the desired sensitivity to fluoride, without the need for a special mutant to be ordered. This fits with our goal for our kit to be a standard which is widely compatible.

For reference, the protocols we used can be found [http://2012.igem.org/Team:Cambridge/Protocolshere].

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