Team:BGIC China/Proof
The Proof of Concept
Design:
Our original design includes two plasmids, one acts as a sensor module (Fig.1), another acts as an signal amplification module (Fig.2).
The sensor plasmid contains pCpxP promoter which is inducible by various carbon sources including glucose, glycerol and citrate acid. Our experiment data also proved that the increase in pH could enchance the expression of pCpxP.
Fig.1 Sensor plasmid (pLV1C3-pCpxP-LacZW-Cre)
Fig.2 Signal amplification plasmid (pET28a-LSL-LacZW-Cre)
Sensor plasmid uses pCpxP promoter to control the expression level of the downstream genes---genes coding for LacZ and Cre. Cre is a recombinase which recognise loxp sites and recombine the target gene based on different directions of two loxp sites which flank the target gene. When Cre recombinase has been expressed from the sensor plasmid, it will cut down two B0015 terminators on the signal amplification plasmid and recombine the plasmid (Fig.3).
Fig.3 Plasmid recombination by Cre at loxp sites
When the B0015 terminators downstream of T7 promoter have been cut off, genes coding for LacZ and Cre could be expressed continuously given that IPTG is in excess supply. Chain reaction will be trigger because recombined signal amplification plasmid itself could express Cre recombinase that cut off B0015 terminators on intact signal amplification plasmids. Thus, the amount of LacZ will be accumulated in an exponential rate and the expression of LacZ will be sustained even pCpxP inducers are used up.
Experiment:
