Best New Part

Our parts offer an accessible method for monitoring putrescine with E. coli biosensors while leveraging hydrogels as a potential avenue for long-term deployment.

PuuR repressor and pVeg1 promoter-New Parts

Parts List

Registry Number	Description
BBa_25087KBX.	PuuR is a transcriptional repressor protein from Escherichia coli that regulates the puu operon involved in putrescine metabolism. PuuR binds to the operator region of puuO, blocking transcription of downstream genes. When intracellular or exogenous putrescine binds to PuuR, the protein undergoes a conformational change that decreases its DNA binding affinity, releasing the operator and permitting transcription.
BBa_25IGLSH4.	pVeg1 is a strong constitutive promoter from B. subtilis.

In order to measure putrescine using whole-cell biosensors, we created a new basic part, PuuR. This basic part encodes for the coding sequence for the PuuR repressor protein. This protein binds to the PuuO operator sequence, repressing transcription of downstream genes. However, in the presence of putrescine, the repressor will bind to the small biogenic amine and change conformation, detaching from the operator sequence and allowing for transcription of the gene.

We measured the performance of this repression using the pHyb(1B) synthetic promoter from Selim et al. [1], which contains two recognition site variants for PuuR. We have also adapted this promoter sequence into a basic part for future iGEM use. The repression activity was monitored using eGFP in the expression circuit below. We showed that this repressor-promoter pair was highly sensitive to putrescine, producing a measurable signal at concentrations of 10mM.

Repression Monitoring Cassette

To monitor the performance of our repressor-inducer circuit, we placed the PuuR coding sequence under the control of a constitutive T7 promoter. We also inserted the mCherry coding sequence with its own RBS just downstream of the PuuR sequence and before the terminator. Although not equal to the amount of repressor in the cell, this mCherry fluorescence allowed us to have some measure of the expression of the repressor at any time in our testing. The pHyb(1B) promoter was placed before eGFP as our reporter protein.

Characterization

Our full experimentation of our New Part can be reviewed on the Experiments page.

Future Directions

In the future, we will investigate swapping the T7 promoter for a different constitutive promoter and also designing a baseline control construct to give a stronger measure of background signal specific to our design. These actions will both serve to help further quantify the efficacy of the PuuR repressor protein.

See our lab notebook page here for more detailed future directions.

Disclaimer:

After further investigation, we uncovered that our backbone had additional promoters which may have influenced our experimental characterization results of this part, PuuR. These promoters are:

1. a LacUV5 promoter upstream of the PuuO synthetic promoter.
2. a reverse T7 promoter downstream of the mCherry CDS.

We are currently working towards modifying our genetic design to exclude these promoters from the construct.

pVeg1 Characterization

For BostonU 2025 characterization, EGFP was put under expression of pVeg1 in NEB® 5-alpha E. coli. See the composite part here: BBa_25IGLSH4. The key contribution of this characterization is the performance of the promoter in varying concentrations of agar hydrogels.

pVeg1 is a variant of the pVeg promoter (BBa_K1819009) engineering by Guiziou et al. (2016). [2]

This composite part generated a strong constitutive GFP signal that served as our positive control for our hydrogel experimentation. Additionally, we aimed for this part to act as our proof of concept for our fluorescence detection system. For details on experimentation and characterization, please refer to our registry page.

References

[1] Selim, A. S., Perry, J. M., Nasr, M. A., Pimprikar, J. M., & Shih, S. C. C. (2022). A synthetic biosensor for detecting putrescine in beef samples. ACS Applied Bio Materials, 5(11), 5487–5496. https://doi.org/10.1021/acsabm.2c00824

[2] Guiziou, S., Sauveplane, V., Chang, H.-J., Clerté, C., Declerck, N., Jules, M., & Bonnet, J. (2016). A part toolbox to tune genetic expression in Bacillus subtilis. Nucleic Acids Research, 44(15), 7495–7508. https://doi.org/10.1093/nar/gkw624