Difference between revisions of "Part:BBa K3771099"

Revision as of 12:46, 18 October 2021

PsoxS-Riboj-sfGFP-J23101-SoxR

Description

This composite part consists of promoter P_soxS Mutant 3 (BBa_K2862010), genetic insulator RiboJ (BBa_K1679038), sfGFP (BBa_K1321337), constitutive promoter J23101 (BBa_J23101), and the soxR gene (BBa_K223000). The concept of this composite part comes from the 2018 Imperial College London iGEM team. The soxR and the soxS gene are components of the soxRS regulon, which is important for E. coli to sense and respond to the oxidants. When SoxR protein is fully oxidized, it becomes a powerful transcription activator of the soxS promoter (P_soxS) (up to 100-fold), leading to the expression of the downstream gene^[1]. RiboJ is a genetic insulator that can increase the expression of insulated genes. sfGFP serves as a reporter to evaluate the strength of P_soxS-Riboj.

Usage and Biology

This composite part was ligated with the pSAA vector and transformed into E. coli. We conducted colony PCR to verify whether E. coli uptake the correct plasmid. The size of the PCR product was as expected.

Fig. 1. The electrophoresis result of colony PCR. M: Marker; Lane 1: pSAA-P_soxS-RiboJ-rcp-J23101-soxR (1103 bp); Lane 2, 3: pSAA-P_soxS-RiboJ-sfgfp-J23101-soxR(998 bp).

With its activator, SoxR, the promoter strength of P_soxS-Riboj under oxidative stress, is determined by the expression level of sfGFP, which is their reporter. While using paraquat (PQ), which is a commonly used agent to induce oxidative stress for bacteria, as the inducer, the intensity of P_soxS-Riboj shows no significant difference under different concentrations of paraquat (Fig. 2).

Fig. 2. Relative fluorescence intensity of P_soxS-Riboj after 4.5-hour incubation with paraquat in various concentrations.

We compared the relative fluorescence intensity of sfGFP expressed by different promoters under the stimulation by different concentrations of paraquat. As expected, sfGFP expression level of P_soxS with its activator, SoxR (BBa_K37710XX), is highly activated under oxidative stress and has the highest gene expression level compared to P_skatG (BBa_K37710XX), P_soxS (BBa_K37710XX) solely, and this composite part. The sfGFP expression by this composite part was quite low (Fig. 3).

Fig. 3.Comparison of different promoter strengths by relative fluorescence intensity after 4.5-hour incubation with paraquat in various concentrations.

References

1. Pomposiello PJ, Demple B. Redox-operated genetic switches: the SoxR and OxyR transcription factors. Trends Biotechnol. 2001;19(3):109-114. doi:10.1016/s0167-7799(00)01542-0

Sequence and Features