Team:HKUST-Rice/Nitrate Sensor PyeaR
Nitrate sensor - yeaRp
Nitrate as a Macro-nutrient
Nitrate is an essential nutrient that involves in multiple roles in plant growth and reproduction.For example, it provides nitrogen that plants need for producing amino acids and nucleic acids (DNA and RNA). Second, it is a component of chlorophyll and therefore essential for photosynthesis. It is also the basic element of plant and animal proteins, and is important in periods of rapid plant growth. Lack of nitrogen will lead to stunted growth and yellowing of leaves, etc.
Result obtained
Responsive range of promoter characterization in Luria Broth (LB)
After we have obtained the quantitative results on GFP signal intensity using an EnVision multilabel reader, we processed the data with relative fluorescence level (in OD600) against nitrate concentration.
We expected to obtain a result that, under low nitrate concentration, the Relative Fluorescence Unit (RFU) will be low and will increase according with increasing nitrate concentration.
From the results obtained, the relative fluorescence level increases by 7.21 folds between 0mM and 10mM concentration of nitrate. Furthermore, a plateau was shown from 10mM nitrate concentration point. This result obtained is expected as according to previous works by Edinburgh iGEM 2009 and BCCS-Bristol iGEM 2010, the dynamic range of yeaRp was from 0-10mM nitrate concentration.
After obtaining the results of yeaRp promoter response behavior in the concentration of 0-50mM nitrate, we can see that between 0-10mM nitrate concentration, the fluorescence signal increases sharply, as a results, another characterization was done focusing on the dynamic range of the promoter, 0-10mM.
Dynamic range characterization in Luria Broth (LB)
After we have obtained the quantitative results on GFP signal intensity using an EnVision multilabel reader, we processed the data with relative fluorescence level (in OD600) against nitrate concentration.
We expected to obtain a result that, under low nitrate concentration, the Relative Fluorescence Unit (RFU) will be low and will increase according with increasing nitrate concentration.
From the results obtained, the relative fluorescence level increases by 4.23 folds between 0mM and 10mM nitrate concentration. Moreover, it shows an upward slope from 0mM to 6mM nitrate concentration. At concentration point 8mM nitrate, it shows a downward slope and then rise again at 10mM nitrate. This result obtained is unexpected as according to previous work by BCCS-Bristol iGEM 2010, a continuous upward slope was obtained from 0mM to 9mM nitrate concentration. The discrepancy in the obtained result and the reference result could be due to use of different bacterial strain, since the strain used in BCCS-Bristol iGEM 2010 was different from ours, the behavior of the promoter may be different.
Responsive range of promoter characterization in M9
After we have obtained the quantitative results on GFP signal intensity using an EnVision multilabel reader, we processed the data with relative fluorescence level (in OD600) against nitrate concentration.
We expected to obtain a result that, under low nitrate concentration, the Relative Fluorescence Unit (RFU) will be low and will increase according with increasing nitrate concentration.
From the results obtained, the relative fluorescence level increases by 4.37 folds from 0mM and 2000μM nitrate concentration, and a plateau was shown from 500μM nitrate concentration point.
After obtaining the results of yeaRp promoter response behavior in the concentration of 0-2000μM nitrate, we find that the relative fluorescence level increases sharply between 0-500μM concentrations of nitrate. As a result, another characterization was done focusing on the dynamic range of the promoter, 0-500μM.
Further Improvements
We were concerned with the endogenous nitrate will affect the sensitivity of the promoter, so, we have designed a method to reduce the endogenous noise.
With araBADp as an inducible promoter, we aimed to find the concentration of arabinose that can reduced the most amount of endogenous noise, so that the promoter can be more sensitive.
As yeaRp promoter is regulated by Nar system and NsrR protein, by the overexpression of nsrR gene, the endogenous nitrate titrate against excess NsrR protein, so that nitrate could not drive the transcription of yeaRp promoter.When there is nitrate in the environment, the amount of nitrate is enough to relieve the repression from the Nar system and NsrR protein, as a result, with the overexpression of nsrR gene, we expected to obtain a result that, the endogenous noise will be lowered, in which the relative fluorescence level at 0mM concentration of nitrate can be lowered to near 0.