Part:BBa_K4907132

I0500-B0034-Mn-SOD-B0015

Biology

The partial reduction of oxygen leads to the formation of various reactive oxygen species (ROS). Superoxide anion radical (O₂•−) is usually the first ROS to be generated. The efficiency of enzymatic reactions decreases at low temperature, leading to the accumulation of intracellular peroxides and causing damage, which is one of the mechanisms of bacterial cold injury. SODs are the main antioxidant enzyme families in organisms. They are considered as the first defense line against oxidative stresses due to their function of converting O₂•− to H₂O₂ and H₂O (1,2). Mn-SOD, a subtype of SOD, utilizes manganese (Mn) as a cofactor, existing in the form of a homotetramer. It is located within the mitochondria of aerobic cells. The expression of Mn-SOD gene in several insect species increased their survival at low temperature (3).

Usage and design

To improve the survival of the engineered bacteria at low temperature, we decide to overexpress Mn-SOD to clear ROS and increase resistance of bacteria to cold stress. We used both BBa_I0500 and BBa_B0034 to construct the expression circuit and obtained the composite part BBa_K4907132 (Fig. 1), which was assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmid was transformed into E. coli DH10β, then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.

Fig. 1 Gene circuits of BBa_K4907132.

Characterization

Agarose gel electrophoresis (AGE)

When constructing this circuit, colony PCR and gene sequencing were used to verify that the transformants were correct. Target bands (2295 bp) can be observed at the position between 2000 bp and 3000 bp (Fig. 2).

Fig. 2 DNA gel electrophoresis of the colony PCR products of BBa_K4907132_pSB1C3.

Antioxidant activity assay by the Oxford cup method

We used Oxford cup antibacterial experiments to observe and compare the antibacterial circle diameter of control strains and experimental strains containing BBa_K4907132 at hydrogen peroxide concentrations of 0, 25, 50, 75, 100 mM respectively. The significant difference (Fig. 3) suggested that the experimental group formed a smaller antibacterial circle at 50 mM hydrogen peroxide. BBa_K4907132 improves the bacterial resistance to peroxides.

Fig. 3 Quantitative diameters of the inhibited zones in histograms.

Survival level of the Mn-SOD-overexpressed DH10β under cold stress at −4 °C

We subjected both control and experimental group to shaking cultivation at -4 °C for 8 hours. We measured the OD₆₀₀ values immediately upon removal and after 1 hour of recovery on a shaking incubator at 37 °C. It can be observed that the overexpressing Mn-SOD strain exhibited significantly enhanced colony growth after one hour of recovery (Fig. 4). BBa_K4907132 demonstrates a protective effect against low-temperature stress on the strains.

Fig. 3 Survival level of bacteria under −4 °C.

Reference

1. N. B. Ackerman, F. B. Brinkley, Oxygen tensions in normal and ischemic tissues during hyperbaric therapy. Studies in rabbits. Jama 198, 1280-1283 (1966).
2. J. M. McCord, I. Fridovich, SUPEROXIDE-DISMUTASE - THE 1ST 20 YEARS (1968-1988). Free Radical Biology and Medicine 5, 363-369 (1988).
3. Y. I. Kim et al., Modulation of MnSOD protein in response to different experimental stimulation in Hyphantria cunea. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology 157, 343-350 (2010).

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961