mRFP production over time

First of all in the characterization of this part, mRFP fluorescence production was measured over time. To accomplish that, a 100 ml culture of EColi DH5alfa carrying a plasmid with a mRFP generator under arsenite sensitive promoter (Bba_K1106003) was grown at 30°C until they reach OD=0.4 (600nm). At this point arsenite was added (1000ppb of arsenite final concentration) and fluorescence was measured every 30 minutes for 8 hours with a fluorimeter at 584 nm excitation peak and 607 nm emission peak. All these data was normalized by the culture density measured by OD.

As it is shown in the figure below, in the presence of arsenite (1000 ppb) a typical transcriptional induction is observed over time. There is a lag of 3 hours in the mRFP produciton after adding arsenite.

Naked eye discernibility range of mRFP production by arsenite inducible promoter

The objective of this assay is to figure out whether bacteria that get colored in the presence of arsenic, reach saturation over time when induced with different arsenite concentrations and if the colour intensity is distinguishable between each other at naked eye. To achieve this, E. Coli DH5alfa Bacteria harbouring a plasmid that encodes mRFP generator under arsenite promoter (Bba_K1106003), were grown with different arsenite concentration ( 0, 10, 50, 200 and 1000ppb). 1ml aliquots were taken every 12 hours and were centrifuged at 10.000rpm for 5 minutes. Pictures of the pellets were taken in order to compare colour difference at naked eye. mRFP fluorescence was also measured with a fluorimeter at X nm for excitation and X nm for emission.

As it can be seen in the pictures below, the colour production can be clearly distinguish between different arsenic concentrations after 24 hours of induction and between the higher arsenite concentrations only. It can also be noticed that over time the production grows and that after xxxtime the difference between 200 and 1000ppb is not clear.

So, in order to accelerate the develop of colour and its quantity at lower arsenite concentrations but avoiding saturation in all the samples to keep the difference observable at naked eye. We concluded that a signal amplification system has to be added but also with some switch to stop the production.