What’s that smell? E. coli’s fine nose for ammonia
Supervisor:Fred Boogerd / Rodolfo Garcia (room G226e)
This single MSc-project (project III) has two well-defined subprojects A and B.
Subproject A: The glnKamtB operon codes for the ammonia sensor-transporter AmtB and its associated regulatory protein GlnK. The latter protein is able to fully block the ammonia transporter. The inactivation occurs within seconds and in response to micromolar changes in the extracellular ammonium concentration. However, the conditions for expression of the glnKamtB operon are poorly understood. Also it is unknown whether the operon plays a role in chemotaxis to ammonia. During growth with excess ammonium as the N-source, neither AmtB nor GlnK are being formed. Only when ammonium was reduced to low levels, AmtB and glnK expression became apparent. However, it remains unclear what the treshold value for ammonium is below which the operon is expressed. Surprisingly, the operon is also transcribed when E. coli grows in a medium with glutamine, proline, or arginine as the single N-source. Also after a period of nitrogen starvation, the operon is expressed. In summary, you will study the conditions for expression of the operon and its relation to chemotaxis.
Subproject B: When the conditions for expression of the glnKamtB operon have been elucidated for media with low levels of ammonium, an intriguing follow-up question is to what extent AmtB-mediated ammonium transport controls the growth rate under this condition. To be able to answer this question, the amtB gene will be cloned in an IPTG-inducible expression vector. The vector will then be inserted in an amtB-negative mutant of E. coli. The expression of the transporter can be carefully modulated in this mutant strain by varying the IPTG concentration. The specific growth rate will then be measured as a function of the expression of the AmtB transporter. Using Metabolic Control Analysis, the control of the transporter on the growth rate around wild type level of the transporter will be determined.
