Team:Aix-Marseille/Contribution
To measure rhamnose production we designed a rhamnose biosensor using an existing biobrick from Paris_Bettencourt team 2012 (BBa_K914003). During our deep bibliography mining about our biosensor we found 3 articles bringing new knowledge to better characterize this biobrick. So, we add recent literature information into the biobrick page, as a contribution to the characterization of the BBa_K914003 biobrick.
The PrhaBAD promoter is an inducible promoter often used in synthetic and molecular microbiology1. This promoter controls the expression of the rhaBAD operon involved in the metabolism of L-rhamnose1.
L-rhamnose metabolism relies on the following genes:
- rhaT, which encodes for a symporter proton-rhamnose1
- the rhaBAD operon, which encodes for proteins that metabolize L-rhamnose1
- the rhaST operon, which encodes for 2 transcriptional activators involved in the regulation of rhaBAD expression.
Rhamnose is firstly imported by RhaT1. When L-rhamnose enters into the cell, it binds to the RhaR transcriptional factor, which is constitutively produced at low levels1. This binding induces the autoinduction of rhaSR expression1. Then, RhaS inducer will bind and activate PrhaT and PrhaBAD promoters1. In parallel, RhaS complexed to L-rhamnose autoregulates negatively its own expression1.
In parallel, RhaS complexed to L-rhamnose autoregulates negatively its own expression.
This system has inspired different biobricks conceptions. Among them, the iGEM team Paris Bettancourt 2012 cloned the PrhaBAD promoter from Escherichia coli in a plasmid to control the expression of a target gene. When used in E. coli, addition of L-rhamnose to the media, allows a dose-dependent activity of the promoter and the system is working thanks to the chromosomic rhaS coding for the transcriptional activator of the PrhaBAD promoter.
This system is very useful. It has the advantage to allow high expression while being locked without the L-rhamnose inducer, it is dose-dependent and the inducer is non-toxic2. However, the induction is transient due to the metabolism of L-rhamnose3. Those parts could be useful to all teams who want to use the PrhaBAD promoter.
The bibliography we read in order to create a biosensor for the rhamnose to assess if our strains produce well this monosaccharide, taught us several things about the PrhaBAD promoter:
- A biosensor for rhamnose gives a stronger signal if a constitutive rhaS gene is added to the plasmid1
- Using a plasmid with a constitutive rhaS allows the use of biobricks in bacteria which don’t express this gene in their chromosome, like cyanobacteria for example2. In fact it allows to decoupled rhaS to the rhaR regulation.
- We can have a response on/off in a rhaB strain mutant.
- Use a E.coli rhaB mutant allows it to have a response independent of the rhamnose metabolism3. This is useful because E.coli metabolize the L-rhamnose and so decrease its quantity with time3.
- Using a rhaB/rhaT mutant bacteria allows to have a real dose dependent induction which is independent of the rhamnose metabolism3. In fact, without this double mutation the production is transient because of the metabolism and so not really dose dependent3. So, this mutant inactivates the L-rhamnose metabolism and the active transport of L-rhamnose (not the passive) to solve this problem and allow a more accurate response3.
- L-mannose is a non-metabolizable analogue able to bind RhaS and induces efficiently the PrhaBAD promoter without being metabolized by E.coli1. It avoids having a transient response of the system but has the same advantages that the L-rhamnose1 .
You can find our contribution here
- Kelly et al, 2016 Synthetic Chemical Inducers and Genetic Decoupling Enable Orthogonal Control of the PrhaBAD Promoter
- Kelly et al, 2018 A Rhamnose-Inducible System for Precise and Temporal Control of Gene Expression in Cyanobacteria
- Hjelm, 2017 Tailoring Escherichia coli for the L‑Rhamnose PBAD Promoter-Based Production of Membrane and Secretory Proteins