Difference between revisions of "Part:BBa K3771096"

Latest revision as of 13:22, 19 October 2021

SoxR-PsoxS-CDO1-6xHis

Description

This composite part consists of the soxR gene (BBa_K3771100), promoter P_soxS (BBa_K3771048), and the cdo1 gene (BBa_K3771041), which encodes L-cysteine dioxygenase (CDO1). Together the whole part is used to express CDO1 to produce taurine under oxidative stress. 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]. The L-cysteine dioxygenase is an enzyme that catalyzes the conversion of L-cysteine into L-cysteine sulfinic acid (cysteine sulfinate). In order to use anti-polyhistidine-tag antibodies to detect the production of CDO1 by western blot, we add 6xHis-tag at the C-terminal of CDO1.

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. The part has been confirmed by sequencing and has no mutations.

Fig. 1. The electrophoresis result of colony PCR. M: Marker; Lane 1: pSAA-soxR-P_soxS-sfgfp (1050 bp); Lane 2, 3: pSAA-soxR-P_soxS-cdo1-6xHis (936 bp).​

After the overnight incubation of E. coli with our plasmid, we diluted the bacteria culture and measured OD₆₀₀ once in a while. Until OD₆₀₀ reached 0.5, we added 0.5 mM paraquat, which served as an oxidative stress inducer, into the culture. We collected 1 ml culture each from the control group and the paraquat group at 2, 4, 6 hours after paraquat was added. Afterward, we conducted SDS-Page and western blot to confirm paraquat was added. Afterward, we conducted SDS-Page and western blot to confirm whether CDO1 expressed successfully under oxidative stress with the regulation of SoxR and P_soxS. With the presence of SoxR, the expression level of CDO1 in the paraquat group is high enough to be detected in western blot, but still insufficient to be seen on SDS-Page (Fig. 2 & 3).

Fig. 2. The SDS-Page result. CDO1 (~22 kDa); –: control; PQ: 0.1 mM paraquat.

Fig. 3. The western blot result. CDO1 (~22 kDa); –: control; PQ: 0.1 mM paraquat.

References

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