Team:UPF CRG Barcelona/Measurement
MEASUREMENT
Real time measurement of promoter activation or gene expression is not an easy task. Usually, specific technologies are needed and they only obtain discrete data. Furthermore, many current real time gene expression measurement systems coupled to reporter proteins (RFP, YFP, GFP…) are subject to leakage or noise. Here, we have developed a simple genetic circuit which aims to modulate the signal caused by the expression of our construct. This circuit is composed of the concatenation of a luxR/plux system followed by the promoter being monitored.
luxR gene and plux promoter can be found naturally in bacteria. They are involved in the process known as quorum sensing, where a bacterial population acts collectively using these two pieces and an inductor: lactone.
Lactone is the molecule that allows for the binding between LuxR and plux. This binding activates the promoter and therefore, makes expression possible. Hence, the activation rate depends directly on the amount of lactone available. In other words, lactone is the modulator of the system.
Consider a generic construct composed by a constitutive promoter, A, an RBS, B, and a gene that we want to express, C. The way in which we apply our system is inserting a RBS, LuxR, a terminator and plux promoter following this order between B and C. This way, if lactone is not introduce into the system, there will be no expression of C. On the contrary, when a high concentration of lactone is introduced in the system it will behave as in normal expressing conditions.
Now imagine that promoter A is not constitutively activated. In this context we will consider three cases:
- High activity of promoter A: A high activity of the promoter means a high production of LuxR to activate plux, but as it has been explained,it has no effect without lactone . In the presence of low quantity of lactone, only a small percentage of LuxR will be able to bind plux, so we would be reducing the gene C expression compared with the original construct.
- Low activity of promoter A: In low quantities of lactone, added to a low quantity of LuxR, the probabilities to have a link to activate plux also reduce the expression of C compared with A This can be applied to eliminate or reduce the leakage of promoter A.
It is important to take into account the assumption that the two RBS in the example before have equal strength. If this strength is modulated, the system’s response will also be modified. RBS strength directly influences the amount of lactone needed to ensure a certain level of expression of C. This way, the weaker the RBS strength, the higher amount of lactone will be needed.
Taking this into account , we have developed a system that can successfully be used as a way to measure promoter activation or gene expression. However, different approaches need to be considered.
Our team has implemented the aforementioned system using RFP as a reporter gene. This fluorescent protein is suitable to our application as a proof of concept. However, it has a very long time of expression and degradation. In other words, we have a delay in the report of LCFA uptake and when expression is stopped in our system (RFP can remain several hours in the medium even if it is no longer being expressed).
A faster reporter protein, the superfolder GFP, with a very short time of expression and degradation would give a more accurate measure of the ON/OFF changes of a system.
Figure 1 | For these measures, the Lactone concentration is 10-7. Here is shown how the LuxR-plux system increases the response compared to the pFadBA promoter