Coding

Part:BBa_K4907024

Designed by: Yannan You   Group: iGEM23_XMU-China   (2023-09-13)
Revision as of 11:54, 12 October 2023 by Yxycf (Talk | contribs) (→‎Agarose gel electrophoresis (AGE))

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katGE

Biology

Mn-SOD

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 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 the Mn-SOD gene in several insect species increased their survival at low temperature (3).

KatG

KatG is a bifunctional enzyme with both catalase and broad-spectrum peroxidase activity. It can convert H2O2 to H2O and O2, which can promote bacterial growth at low temperature to some extent. Furthermore, it also performs NADH oxidase, INH lyase, and isonicotinoyl-NAD synthase activities (1).

T7 RBS

T7 RBS is a ribosome binding site on the genome of the T7 phage, which is capable of recruiting ribosomes in engineered E. coli.

Usage and design

Through previous experiments, we have proved that Mn-SOD (BBa_K4907023) can improve the low temperature stress resistance of engineered bacteria. Mn-SOD can catalyze active oxygen species in bacteria to generate hydrogen peroxide. Considering that hydrogen peroxide is also harmful to bacteria, we hope to introduce KatG as a catalytic enzyme to degrade hydrogen peroxide catalyzed by Mn-SOD. We use BBa_K4907132, BBa_K4907041, BBa_K4907024 to construct BBa_K4907134_pSB3K3 via the structure of polycistrons to verify this hypothesis. The constructed plasmid was transformed into E. coli BL21(DE3), then the positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing.

Fig. 1 Gene circuit of Mn-SOD

Characterization

Agarose gel electrophoresis (AGE)

When building this circuit, colony PCR was used to certify the plasmid was correct. We got the target fragment-1198 bp (lane K4907110).

Fig. 2 The result of colony PCR. Plasmid BBa_K4907134_pSB3K3

We transferred BBa_K4907134_pSB3K3 (as a positive control group), BBa_K4907132_pSB3K3 (as the experimental group), and BBa_K4907118_pSB1C3 respectively into E. coli BL21(DE3) and the correct dual-plasmid transformants were selected by chloramphenicol and kanamycin. Further, the OD600 value and fluorescence intensity of the above bacteria were recorded at 4 °C. For more details of the experiment please see our Experiment. As shown in Fig. 3, we found that bacteria containing katG did not show stronger growth ability and protein expression ability as expected. We believe that this may be the effect of Mn-SOD is too strong and the effect of katG is relatively not significant.

Fig. 3 The comparison of normalized fluorescence intensity between BBa_K4907134 and BBa_K4907132

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).
4. B. L. Triggs-Raine, B. W. Doble, M. R. Mulvey, P. A. Sorby, P. C. Loewen, Nucleotide sequence of katG, encoding catalase HPI of Escherichia coli. Journal of Bacteriology 170, 4415-4419 (1988).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 193
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 193
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 193
    Illegal BglII site found at 228
    Illegal BglII site found at 1163
    Illegal BglII site found at 1290
    Illegal BamHI site found at 1080
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 193
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 193
  • 1000
    COMPATIBLE WITH RFC[1000]


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