Part:BBa_K3771094

PsoxS-CS-6xHis

Description

This composite part consists of promoter P_soxS (BBa_K3771048) and the cs gene (BBa_K3771012), which encodes L-cysteine sulfonic acid synthase (CS). It is used to express CS to produce taurine under oxidative stress. The soxS gene is one component of the soxRS regulon, which is important for E. coli to sense and respond to the oxidants. The soxS promoter (P_soxS) is stimulated by oxidative stress, leading to the expression of the downstream gene. The L-cysteine sulfonic acid synthase (CS) is an enzyme that catalyzes the conversion of O-phospho-L-serine and sulfite into L-cysteine sulfonic acid (L-cysteate) and phosphate. In order to use anti-polyhistidine-tag antibodies to detect the production of CS by western blot, we add 6xHis-tag at the C-terminal of CS.

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-P_soxS-sfgfp (2691 bp); Lane 2, 3: pSAA-P_soxS-cs-6xHis (3240 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 CS expressed successfully under the regulation of P_soxS by oxidative stress. The expression level of CS is too low to be seen on SDS-PAGE without its activator, SoxR (Fig. 2); nevertheless, the result of western blot shows more CS production in the paraquat group (Fig. 3).

Fig. 2. The SDS-PAGE result. CS (~46 kDa); –: control; PQ: 0.5 mM paraquat.

Fig. 3. The western blot result. CS (~46 kDa); –: control; PQ: 0.5 mM paraquat.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

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