Here is a summary table of all the basic and composite parts the team AshesiGhana used this year. You will find information below, such as the part's purpose and functions, the part type and where it can be found on the registry. Also, the team created a basic part and other composite parts for sensing Fe, Au and As. Below is our team's new basic and composite part and an extensive explanation of what these parts do.
ArsR is a regulatory protein that binds to the promoter region of arr, ars, and specific arsM operons and inhibits their transcription unless arsenite is present. This tight regulation of gene expression by the ars operon promoter (Pars) can be used to develop arsenic biosensors. The ParWT basic part our team created is a constitutive promoter that binds with arsenite when it is present and allows for transcription to occur.
The UV in the kill switch composite part is an inducible promotor regulated by UV irradiation. The promoter is triggered in the presence of UV light, which allows the rest of the genetic circuit to be transcribed. The lysozyme from enterobacteria phage T4 degrades the peptidoglycan layer. We successfully combined T4 endolysin (BBa_K112806) with a UV inducible promoter (BBa_I765001), a ribosome binding site (BBa_J61100) and a terminator (BBa_B0010). This composite part comes together to form a functional suicide mechanism.
The ferric inducer composite comprised a constitutive promoter, Pcat (BBa_I14033), which has a medium transcription and it is coded for chloramphenicol resistance, which induces the production of the FUR protein. The FUR (BBa_K1122666) protein is a repressor for the PAceB (BBa_K1163101), and it is always produced. Hence, in the presence of Fe (2+), the protein produced by the FUR binds with the Fe ions, repressing the PAceB. In between the fur and the PAceB promoter is the T7Te terminator (BBa_B0012),. This is a transcription terminator for the E. coli RNA polymerase that is a promoter in the reverse direction. The pTET (BBa_K2135002) is a constitutive promoter that TetR represses. Hence, the TetR protein is not transcribed in the presence of Fe ions, allowing Ptet to transcribe the reporter gene eforRed (BBa_K1073023). The chromoprotein eforRed expresses a pink color that the naked eye can see.
The ParsWT is a constitutive promoter with a tight gene expression by the ars operon obtained through literature review. In the presence of arsenite, the ions bind with the ArsR protein (BBa_J33201), allowing for transcription. The reporter gene used in our design is the amilGFP (BBa_K592010); this is a chromoprotein from the coral Acrapora millepora, which naturally exhibits strong yellow color when expressed. In the design, the chromoprotein is readily visible to the naked eye after transcription. The terminator (BBa_B0010) at the end of the genetic sequence ensures that transcription stops after the gene is expressed.
The PgolTS- golS-PgolB (BBa_K1520506) is a composite part acquired from iGEM’s part registry. In the presence of gold ions, the promoter PgolTS-golS-PgolB causes the production of the downstream gene. PgolTS functions as a continuous promoter, generating golS, an inhibitor of PgolB. Once gold ions are combined, inhibitor PgolB will no longer function. Therefore, the CDS after this portion will be expressed when there are gold ions. In this design, the CDS is only amilCP (BBa_K592009), a chromoprotein from the coral Acropora millepora, that naturally exhibits strong blueish colour when expressed. There is a Ribosome Binding Site (BBa_J61100) between the composite promoter and the reporter gene to ensure that reporter gene is effectively translated. Finally, there is a terminator (BBa_B0010) at the end of the sequence to stop the transcription of the genetic circuit.