Design

The aim of the project is to identify the promoters that are activated by the exudates of common crops via the root exudates. Applications of this idea are endless, but as a case study, we have designed a amplification circuit which can in turn be used to improve the efficiency of phosphate solubilization in the soil. Our chassis organism is Bacillus subtilis, a common soil bacterium. As this amplification circuit is going to be activated by the root exudates of particular crops, this phosphate solubilization will take place only in the rhizosphere of the crops of interest and not in the fallow land.

Genetic Amplification Circuit

Description

The construct that we have designed, is theoretically self-sustainable, once activated by the inducer. P₁ is the inducible promoter. G₁ is a sequence of bases that codes for the activator protein which acts as an inducer to the promoter P₂. Promoter P₂, when induced, starts the transcription of G₁ along with a reporter gene, Red Fluorescent Protein (RFP) in this case. As the transcription of G₁ is started again, promoter P₂ is continuously turned on, which makes this construct self-sustainable.

Promoter P₁

For experimental purposes, while we have not received the RNA sequencing results, we tried out various inducible promoters for Bacillus subtilis. We were able to get one promoter P_liaI (Part # BBa_823001) from the iGEM 2018 distribution kit, which is a bacitracin inducible promoter for B subtilis. We were not able to get expected results with it.

We also tried to PCR amplify a few inducible promoters from the Bacillus subtilis 168 genome, namely:

1. pMalA: Maltose inducible promoter
2. pXylA: Xylose inducible promoter
3. pMtlA: Mannitol inducible promoter

Gene G₁ and Promoter P₂

These two parts are the soul of the whole construct as these are the ones which make the circuit self-sustainable. Promoter P₂, which is induced by the activator protein coded by the gene G₁ again starts the transcription of the same gene G₁. Hence, when the circuit is activated by the inducible promoter P₁, transcription of G₁ takes place continuously. We found two such parts in the iGEM distribution kit, and tried to build the construct with both of them.

1. 71 Ogr
2. 80 Pag

Reporter Gene

Aim of this amplification is to increase the efficiency of phosphate solubilization. The gene PhoD, in the genomic DNA of B subtilis strain 168 codes for the phosphodiasterase, which helps in assimilation of organic phosphorous in the soil. We were able to extract this gene out of the genomic DNA of B subtilis strain 168 by PCR amplification. But decided to use the reporter gene RFP, readily available in the iGEM Distribution Kit for characterization of our construct for its ease in quantification.