View source for Part:BBa K4907041


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<partinfo>BBa_K4907041 short</partinfo>
===Biology===
====Mn-SOD====
The partial reduction of oxygen leads to the formation of various reactive oxygen species (ROS). Superoxide anion radical (O<sub>2</sub>&bull;<sup>−</sup>) 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 O<sub>2</sub>&bull;<sup>−</sup> to H<sub>2</sub>O<sub>2</sub> and H<sub>2</sub>O (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 H<sub>2</sub>O<sub>2</sub> to H<sub>2</sub>O and O<sub>2</sub>, 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 <i>E. coli</i>.

===Usage and design===
Through previous experiments, we have proved that Mn-SOD (<partinfo>BBa_K4907023</partinfo>) 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 <partinfo>BBa_K4907132</partinfo>, <partinfo>BBa_K4907041</partinfo>, <partinfo>BBa_K4907024</partinfo> to construct BBa_K4907134_pSB3K3 via the structure of polycistrons to verify this hypothesis. The constructed plasmid was transformed into <i>E. coli</i> BL21(DE3), then the positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing.
<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/i0500-b0034-mn-sod-t7-rbs-katge.png" width="400px"></html></center>
<center><b>Fig. 1 Gene circuit of Mn-SOD</b></center>

===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).
<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/134-1.png" width="400px"></html></center>
<center><html><B>Fig. 2 The result of colony PCR. Plasmid BBa_K4907134_pSB3K3 </B></html></center>

We transferred BBa_K4907134_pSB3K3 (as a positive control group), BBa_K4907132_pSB3K3 (as the experimental group), and BBa_K4907118_pSB1C3 respectively into <i>E. coli</i> BL21(DE3) and the correct dual-plasmid transformants were selected by chloramphenicol and kanamycin.
Further, the OD<sub>600</sub> value and fluorescence intensity of the above bacteria were recorded at 4 &deg;C. For more details of the experiment please see our [https://2023.igem.wiki/xmu-china/experiments Experiment]. As shown in Fig. 3, we found that bacteria containing <i>katG</i> did not show stronger growth ability and protein expression ability as expected. We believe that this may be because the effect of Mn-SOD is too strong and the effect of <i>katG</i> is relatively not significant.
<center><html><img src="https://static.igem.wiki/teams/4907/wiki/parts/jincheng/katg.png" width="400px"></html></center>
<center><b>Fig. 3 The comparison of normalized fluorescence intensity between <partinfo>BBa_K4907134</partinfo> and <partinfo>BBa_K4907132</partinfo></b></center>

===Reference===
1.	N. B. Ackerman, F. B. Brinkley, Oxygen tensions in normal and ischemic tissues during hyperbaric therapy. Studies in rabbits. <i>Jama</i> <b>198</b>, 1280-1283 (1966).<br/>
2.	J. M. McCord, I. Fridovich, SUPEROXIDE-DISMUTASE - THE 1ST 20 YEARS (1968-1988). <i>Free Radical Biology and Medicine</i> <b>5</b>, 363-369 (1988).<br/>
3.	Y. I. Kim et al., Modulation of MnSOD protein in response to different experimental stimulation in <i>Hyphantria cunea</i>. <i>Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology</i> <b>157</b>, 343-350 (2010).<br/>
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 <i>Escherichia coli</i>. <i>Journal of Bacteriology</i> <b>170</b>, 4415-4419 (1988).


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===Usage and Biology===

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<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K4907041 SequenceAndFeatures</partinfo>


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===Functional Parameters===
<partinfo>BBa_K4907041 parameters</partinfo>
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