Difference between revisions of "Part:BBa K3771098"

Revision as of 03:52, 18 October 2021

PsoxS-Riboj-RCP-J23101-SoxR​

Description

This composite part consists of promoter P_soxS Mutant 3 (BBa_K2862010), genetic insulator RiboJ (BBa_K1679038), RCP (BBa_K592012), constitutive promoter J23101 (BBa_J23101), and the soxR gene (BBa_K223000).

   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 (PsoxS) (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. RCP serves as a reporter to help us pick up the correct colony. 

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-sfgfp (990 bp); Lane 2, 3: pSAA-P_soxS-RiboJ-rcp-J23101-soxR (1103 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 CSAD expressed successfully under oxidative stress with the regulation of SoxR and P_soxS. With the presence of SoxR, the expression level of CSAD 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. CSAD (~55 kDa); –: control; PQ: 0.1 mM paraquat.

Fig.3. The western blot result. CSAD (~55 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