Design

The project revolves around enabling phosphate solubilization in the soil in presence of specific crops. For this, we wished to construct a genetic circuit which leads to production of a phosphate solubilizing enzyme when induced by the exudates of the crop grown. The circuit should to be able to produce a substantial amount of the phosphate solubilizing enzyme (like alkaline phosphatase) only when induced by the exudates. Additionally, high phosphate solubilization will make it easier for the crop to assimilate phosphorous from the soil. Keeping this in mind, we decided to produce an amplified response of the phosphatase enzyme in presence of specific exudates.

The constructed genetic circuit is a positive-feedback amplification circuit where a transcriptional activator activates a promoter leading to transcription of itself and a target gene, thus further promoting formation of itself. This circuit has been shown to work in a robust manner and provide a higher level of gene expression than a circuit without the amplification function. [1]  We have added an inducible promoter at the start of the construct to first initiate expression of the transcriptional activator which will then initiate the amplification function. This will result in an amplified response of our target gene (phoD) in presence of an inducer, which in our case will be the root exudates of the crops.

Amplification Circuit Design

Description

The designed construct is a positive feedback genetic amplifier which is activated when induced by an external chemical moiety (root exudates in this case). P_X is such an inducible promoter. TA is a transcription activator that activates transcription from the promoter P_TA. Promoter P_TA, when induced, starts the transcription of TA along with a reporter gene, rfp. As the transcription of TA is continuous, promoter P_TA is continuously turned on and the response to the inducer is amplified.

Promoter P_X

P_X is a inducible promoter which is turned on when activated by an external stimulus. Via RNAseq, we wish to identify promoters of genes that are upregulated in Bacillus subtilis 168 in presence of root exudates of various crops. While the results of RNAseq are awaited, a bacitracin-inducible promoter, P_LiaI (Part #BBa_K823001 ) was pulled out from the Parts Registry. We also tried to clone various sugar inducible promoters such as, P_malA (maltose inducible), P_xylA (xylose inducible), and P_mtlA (mannitol inducible), from the genomic DNA of B. subtilis 168. Constitutive promoters for B. subtilis from the Parts Registry were used to check validity of the construct. For detailed results, see results page.

Transcription Activator (TA)

The transcription activator is the most important part of the construct as it activates the promoter which in turn activates transcription of the transcription activator itself. In order to construct the positive feedback amplifier, we required a transcription activator which did not interfere with native gene regulation in neither B. subtilis nor E. coli and acted as an inducer to another promoter as well.
Two such transcription activators were chosen from the iGEM 2018 Distribution Kit.

1. Pag activator from phage PSP3 (Part #BBa_I746351)
2. Ogr activator from phage P2 (Part #BBa_I746350)

For detailed results, see results page.

Promoter induced by transcription activator (P_TA)

P_TA is the promoter which, when induced by the transcription activator, drives the transcription of the output reporter gene along with transcription activator itself. The promoters which could be activated by Pag or Ogr activators were required for the construct. Two such promoters were chosen from the iGEM 2018 Distribution Kit:

1. Promoter PF from phage P2 (Part #BBa_I746360)
2. Promoter PO from phage P2 (Part #BBa_I746361)

As both of these are non-native to the chassis organism, interference with native gene regulation is not expected.

Reporter Gene

The positive feedback circuits leads to the amplification of response to inducers. For the quantification of this response, the RFP reporter was selected as the output for its ease in characterization using measurement of fluorescence and availability in the iGEM 2018 Distribution Kit.

Please click here for detailed documentation of results.

References

1. Nistala, G.J., Wu, K., Rao, C.V. and Bhalerao, K.D., 2010. A modular positive feedback-based gene amplifier. Journal of biological engineering, 4(1), p.4.