Team:UPNAvarra Spain/Design
Design
How to design a bacterial biosensor?
Biosensors are bacteria able detect and respond to different stimulus. To generate a bacterial biosensor, we have to introduce a previously design plasmid into the bacteria we want to modify. In general, this plasmid must contain a sensitive promoter, a RBS necessary for the transcription, the reporter gene and a transcription terminator. In our case, these sequence have been cloned into the pSB1C3 vector and transformed into E. coli cells.
In our project we want to develop different pollutant biosensors, able to detect and quantify those pollutants through a simplistic imaging method. In that case, our reporters are diferent chromoproteins and each contaminant will be associated with a different colour.
In our project we want to develop different pollutant biosensors, able to detect and quantify those pollutants through a simplistic imaging method. In that case, our reporters are diferent chromoproteins and each contaminant will be associated with a different colour.
General Design
Given the detailed background about biosensors existing on iGEM competition, we decided to use already existing parts to construct our biosensors, with the aim of improve the way of pollutant detection and quantification.
For that purpose, we started with an already existing nitrate sensor for our system, design by NEU_China_A in iGEM 2018 (BBa_K2817007). It is composed of the nitrate sensitive promoter PyeaR, a strong RBS, the amilCP blue chromoprotein and a transcriptional terminator (Figure 1). This promoter was firstly used by Edinburgh team in 2009, which is the promoter of the Escherichia coli yeaR/yoaG operon. The usage of the amilCP blue protein (design by the Uppsala-Sweden team in iGEM 2011) behind the PyeaR promoter, let us identify the presence of nitrate without any special equipment. So, in presence of nitrate, bacteria turn into different blue color intensities according to the concentration of nitrate.
Figure 1. Construction of the nitrate biosensor BBa_K2817007 (our twin BBa_K3287000) in the pSB1C3 vector. It consists of the PyeaR promoter, a strong RBS in front of the amilCP chromoprotein and a transcription terminator.
Trying to improve this construction, we designed the same biosensor with a different chromoprotein, amilGFP (design by the Uppsala-Sweden team in iGEM 2011) (Figure 2). With this new reporter gene we are going to be able to better characterize the PyeaR promoter.
Figure 2. Construction of the nitrate biosensor BBa_K3287001 in the pSB1C3 vector. It consists of the PyeaR promoter, a strong RBS in front of the amilGFP chromoprotein and a transcription terminator.
As we were also interested on heavy metal detection, we designed three biosensors in order to detect coper, mercury and cadmium.
Our sensor for coper detection is based on the already existing sensor created by Bielefed_CeBiTec in 2015, consisting of CueR activator and the copper specific promoter copAP. The activator is under the control of a constitutive promoter. The promoter copAP is regulated by the acivator, which binds Cu2+ ions. However, we placed a strong RBS and the chromoprotein amilCP downstream the promoter for a first sight detection (Figure 3).
Figure 3. Construction of the coper biosensor BBa_K3287002 in the pSB1C3 vector. It consists of the CueR activator under the control of a constitutive promoter, a copper inducible promoter copAP (*a portion of the BBa_K1758323 sequence), a strong RBS in front of the amilCP chromoprotein and a transcription terminator.
In case of mercury, we constructed our sensor based on the the mercury dependent mer operon from Shigella flexneri R100 plasmid Tn21, with already existing parts designed by Peking 2010 and Bielefed_CeBiTec 2015. It contains the MerR activator under the control of a constitutive promoter, which regulates the mercury specific promoter pmerT. Unlike the existing sensor, we placed a strong RBS and the chromoprotein tsPurple (design by the Uppsala-Sweden team in iGEM 2011) downstream the promoter for a first sight detection (Figure 4).
Figure 4. Construction of the mercury biosensor BBa_K3287003 in the pSB1C3 vector. It consists of the MerR activator under the control of a constitutive promoter, a mercury inducible promoter PmerT, a strong RBS in front of the tsPurple chromoprotein and a transcription terminator.
Finally, we design a cadmium biosensor using a regulatory sequence previously designed by SCUT team in iGEM 2015. This is a cadmium sensitive promoter, that regulates the expression of the downstream placed efordRed chromoprotein (design by the Uppsala-Sweden team in iGEM 2011). With this construction we will be able to detect the presence of cadmium because bacteria will turn into different pink color intensities according to the concentration of cadmium.
Figure 5. Construction of the cadmium biosensor BBa_K3287004 in the pSB1C3 vector. It consists of the cadmium inducible promoter CadA, a strong RBS in front of the efordRed chromoprotein and a transcription terminator.
For that purpose, we started with an already existing nitrate sensor for our system, design by NEU_China_A in iGEM 2018 (BBa_K2817007). It is composed of the nitrate sensitive promoter PyeaR, a strong RBS, the amilCP blue chromoprotein and a transcriptional terminator (Figure 1). This promoter was firstly used by Edinburgh team in 2009, which is the promoter of the Escherichia coli yeaR/yoaG operon. The usage of the amilCP blue protein (design by the Uppsala-Sweden team in iGEM 2011) behind the PyeaR promoter, let us identify the presence of nitrate without any special equipment. So, in presence of nitrate, bacteria turn into different blue color intensities according to the concentration of nitrate.
Trying to improve this construction, we designed the same biosensor with a different chromoprotein, amilGFP (design by the Uppsala-Sweden team in iGEM 2011) (Figure 2). With this new reporter gene we are going to be able to better characterize the PyeaR promoter.
As we were also interested on heavy metal detection, we designed three biosensors in order to detect coper, mercury and cadmium.
Our sensor for coper detection is based on the already existing sensor created by Bielefed_CeBiTec in 2015, consisting of CueR activator and the copper specific promoter copAP. The activator is under the control of a constitutive promoter. The promoter copAP is regulated by the acivator, which binds Cu2+ ions. However, we placed a strong RBS and the chromoprotein amilCP downstream the promoter for a first sight detection (Figure 3).
In case of mercury, we constructed our sensor based on the the mercury dependent mer operon from Shigella flexneri R100 plasmid Tn21, with already existing parts designed by Peking 2010 and Bielefed_CeBiTec 2015. It contains the MerR activator under the control of a constitutive promoter, which regulates the mercury specific promoter pmerT. Unlike the existing sensor, we placed a strong RBS and the chromoprotein tsPurple (design by the Uppsala-Sweden team in iGEM 2011) downstream the promoter for a first sight detection (Figure 4).
Finally, we design a cadmium biosensor using a regulatory sequence previously designed by SCUT team in iGEM 2015. This is a cadmium sensitive promoter, that regulates the expression of the downstream placed efordRed chromoprotein (design by the Uppsala-Sweden team in iGEM 2011). With this construction we will be able to detect the presence of cadmium because bacteria will turn into different pink color intensities according to the concentration of cadmium.