Team:CIDEB-UANL Mexico/Project/Circuit

The circuit of this project is about a biosensor that will measure the concentration of Arabinose in a simple. The circuit has 3 sections: High concentration, Low concentration and Stand-by (when there is no presence of Arabinose). Each section has a color the High concentration is represented with yellow fluorescence, the Low concentration with red and the Stand-by with green.

The first part is called Stand-by. It is turned on all the time and it indicates that there is no presence of Arabinose in the sample. Thus we need a constitutive promoter that is always working and in this case we have the part R0053 that corresponds to the promoter p22-cll that its position in the DNA plates sent from the MIT is 1-6M. We have to observe a response to know if the promoter is always working. In this case, a reporter will be used. This reporter is a GFP protein that emits green color fluorescence. The following part is a RBS followed by a reporter. In this case, we found a biobrick, K081012 with a location in plates 3-12M, that includes a RBS, a reporter, and a terminator, all together in a vector. What we have to do is to join the constitutive promoter 1-6M with the 3-12M biobrick.

In the High concentration section, we use the pBAD promoter which works with the AraC and acts as a repressor: when there is no Arabinose it represses the DNA transcription and when Arabinose is present it keeps working the pBAD. When Arabinose enters into the bacteria it is joined with the AraC and these 2 molecules joined are the ones that will be quantified. This promoter is found in a biobrick, I0500, which also contains an AraC part. This biobrick has a location 1-14N in the plates. The following part is a RBS, but it has to be compatible with the circuit. We found 3 RBS compatible and we chose the B0034 which is the most recommended of the 3 in the Registry of Standard Biological Parts (http://partsregistry.org/Main_Page) and it has a location 1-2M in the plates. In this section, we need a response that indicates the presence of the Arabinose in high concentration, and this response will be through the YFP reporter. This part is the E0032 with a location 1-6E. Then we need another RBS and another part that will repress the Low concentration section because Low and High concentration work with the same promoter, as we can see in the first image of the whole circuit. The repressor will act when the response of the amount of Arabinose measured is bigger than the response of the low concentration. In this way only the part of High concentration will keep on. The part p0152 with a location 1-10E, contains the RBS, the repressor, and a terminator. This is the last part of the High concentration section.

The parts that compose the Low concentration section are: the promoter pBAD I0500 (1-14N) which is the same of the High concentration, the part I746352 (2-12G) which has a RBS with a phiR73 and it is joined with the p0153 (1-10G). The amount of Arabinose is measured by the pBAD and then the response is increased by the sensitivity tuner. The phiR73 (that is in the 2-12G) receives the amount measured by the pBAD and it sends the signal to the pLL wich is a promoter I746365 (2-14A) which is joined with the part p0151 (1-10C). The p0151 has a RBS and the cI part that multiplies the response received from the phiR73 and it sends it to the part I12006 (2-11J). It also has a terminator. Then the part 2-11J is the promoter that receives the response increased from the cI. It is joined with the part K081014 (3-12O) which has a RBS, the RFP that will give the red fluorescence response and a double terminator. The promoter receives the response increased and with this final response the red color appears. In this final part is where the high concentration’s repressor acts. The part 1-10G has a repressor which turns off the stand-by section of the circuit avoiding to have the 3 colors in the bacteria at the same time.

Here it goes a simple video for demonstration how our circuit works. Hope you enjoy it.