Difference between revisions of "Part:BBa K3983002"

 
 
Line 3: Line 3:
 
<partinfo>BBa_K3983002 short</partinfo>
 
<partinfo>BBa_K3983002 short</partinfo>
  
−
pro-efeB
+
Promoter
 +
=== Profile ===
 +
==== Name: pro-efeB ====
 +
==== Base Pairs: 2107 bp ====
 +
==== Origin: Escherichia coli str. K-12 substr. MG1655 ====
 +
==== Properties: Gene technology for protecting patented bacterial strains ====
 +
 
 +
=== Usage and Biology ===
 +
 
 +
In modern society, more and more people suffer from mild or severe depression. It has been reported that malondialdehyde (MDA) level in the plasma of depressed patients is significantly increased. After receiving conventional antidepressant treatment, the patient's MDA level decreased to the same as that of healthy people. Therefore, researchers believe that oxidative stress may play an important role in the occurrence and development of depression, and the activity of antidepressant therapy may be mediated by improving oxidative stress/antioxidant function. We attempt to express an antioxidant enzyme system (peroxidase gene efeB) in the cell or on the surface of the engineered probiotic bacteria to inhibit the production of malondialdehyde (MDA) by human cells and prevent cell oxidation from causing health damage to the body. So as to prevent or alleviate the condition of depression.
 +
The oxidation rate of peroxisome increases in proportion to the increase of oxygen tension. Especially in the case of high oxygen concentrations, the oxidation reaction of peroxisomes dominates. This characteristic allows peroxisomes to protect cells from the toxic effects of high concentrations of oxygen. efeB reduces the oxidative stress in the cell by competing with mitochondria for oxygen, and reduces the MDA produced during the oxidation of lipids.
 +
 
 +
[[File:T--The Webb Schools--BBa K3983002-figure1.jpg|500px|thumb|center|Figure 1. Action and function of efeB in MDA reduction...]]
 +
 
 +
=== Construct design ===
 +
 
 +
[[File:T--The Webb Schools--BBa K3983002-figure2.jpg|500px|thumb|center|Figure 2. The structure of efeB vector...]]
 +
 
 +
=== BBa_K3983002===
 +
==== Name: pro-efeB ====
 +
==== Base Pairs: 2107 bp ====
 +
==== Origin: Escherichia coli str. K-12 substr. MG1655 ====
 +
==== Properties: Gene technology for protecting patented bacterial strains ====
 +
 
 +
Peroxidase EfeB, originally studied as a substrate of the E. coli "double arginine" translocation system, is the first recognized peroxidase for bacterial dye decolorization. Subsequent studies have shown that EfeB is a peroxidase with heme as a prosthetic group, and has a specific iron transport function.We use E. coli as the starting strain and construct an engineered strain of efeB to explore the effects of efeB on bacterial growth, malondialdehyde concentration, ROS synthesis and antioxidant pathways under different oxidative stress conditions.
 +
 
 +
=== Engineering Success ===
 +
==== Production, purification, and Agarose gel analysis of recombinant efeB ====
 +
 
 +
In order to present the function of the part, the efeB gene was expressed in E. coli under the control of T7 promoter. Then the bacterial cells are collected and crushed, and the efeB enzyme solution is purified by further confirmation by the Agarose gel electrophoresis, which is found in the corresponding protein band of approximately 2.1kb (Figure 3).
 +
 
 +
[[File:T--The Webb Schools--BBa K3983002-figure3.jpg|500px|thumb|center|Figure 3 The result of Agarose gel electrophoresis...]]
 +
 
 +
=== Bacteria Growth ===
 +
Bacteria A contains plasmid A, pUC57-efeB, and the bacteria D contains plasmid D, pUC57. Cultivate them in H2O2 solutions of different concentrations.When the concentration of hydrogen peroxide is 0 mM, the relative biomass of bacteria A and bacteria D are the same. As the concentration of hydrogen peroxide increases to 1mM, the relative biomass of bacteria A increases while the relative biomass of bacteria D remains the same, which shows that without efeB, bacteria is unable to grow when ROS is present. As the concentration of hydrogen peroxide increases to 2mM, the relative biomass of bacteria A decreases by half while the relative biomass of bacteria D decreases to 0mM. This shows that bacteria with the efeB gene is able to survive when the level of ROS increases. As the concentration of hydrogen peroxide increases to 4mM, the relative biomass of both bacteria A and bacteria D are zero. This shows that when the level of ROS is too high, bacteria are unable to survive regardless of the presence of the efeB gene.
 +
 
 +
[[File:T--The Webb Schools--BBa K3983002-table1.png|500px|thumb|center|Table 1. relative biomass percents of bacteria A and D with different concentrations of H2O2 ...]]
 +
 
 +
[[File:T--The Webb Schools--BBa K3983002-figure4.jpg|500px|thumb|center|Figure 4. Histogram of relative biomass of bacteria A and D against
 +
different concentrations of H2O2 ...]]
 +
 
 +
Bacteria A contains plasmid A, pUC57-efeB, and the bacteria D contains plasmid D, pUC57. When the concentration of hydrogen peroxide is 0 mM, the relative biomass of bacteria A and bacteria C are the same. As the concentration of hydrogen peroxide increases to 1mM, the relative biomass of bacteria A increases while the relative biomass of bacteria C remains the same, which shows that without efeB, bacteria is unable to grow when ROS is present. As the concentration of hydrogen peroxide increases to 2mM, the relative biomass of bacteria A decreases by half while the relative biomass of bacteria D decreases to 0mM. This shows that bacteria with the efeB gene is able to survive when the level of ROS increases. As the concentration of hydrogen peroxide increases to 4mM, the relative biomass of both bacteria A and bacteria D are zero. This shows that when the level of ROS is too high, bacteria are unable to survive regardless of the presence of the efeB gene.
 +
 
 +
=== Reference ===
 +
==== [1] McCarter T. (2008). Depression overview. American health & drug benefits, 1(3), 44–51. ====
 +
 
 +
==== [2] Chand SP, Arif H. Depression. [Updated 2020 Nov 18]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK430847/ ====
 +
 
 +
 
 +
==== [3] Wang, J., Wu, X., Lai, W., Long, E., Zhang, X., Li, W., Zhu, Y., Chen, C., Zhong, X., Liu, Z., Wang, D., & Lin, H. (2017). Prevalence of depression and depressive symptoms among outpatients: a systematic review and meta-analysis. BMJ open, 7(8), e017173. https://doi.org/10.1136/bmjopen-2017-017173 ====
 +
 
 +
==== [4] Bueno-Notivol, J., Gracia-García, P., Olaya, B., Lasheras, I., López-Antón, R., & Santabárbara, J. (2021, January 1). Prevalence of depression during the COVID-19 outbreak: A meta-analysis of community-based studies. International Journal of Clinical and Health Psychology. DOI: 10.1016/j.ijchp.2020.07.007. ====
 +
 
 +
==== [5] U.S. Department of Health and Human Services (HHS). (n.d.). Major Depression. National Institute of Mental Health. https://www.nimh.nih.gov/health/statistics/major-depression#part_155720.  ====
 +
 
 +
==== [6] Ferguson, J. M. (2001, February). SSRI Antidepressant Medications: Adverse Effects and Tolerability. Primary care companion to the Journal of clinical psychiatry. Doi: 10.4088/pcc.v03n0105 ====
 +
 
 +
==== [7] Jiménez-Fernández, S., Gurpegui, M., Dí¬az-Atienza, F., Pérez-Costillas, L., Gerstenberg, M., & Correll, C. U. (2021, March 1). Comparison of ODD vs Healthy Controls. Psychiatrist.com. dx.dot.org/10.4088/JPC.14r09179. ====
 +
 
 +
==== [8] Rowe, L. A., Degtyareva, N., & Doetsch, P. W. (2008). DNA damage-induced reactive oxygen species (ROS) stress response in Saccharomyces cerevisiae. Free radical biology & medicine, 45(8), 1167–1177. https://doi.org/10.1016/j.freeradbiomed.2008.07.018 ====
 +
 
 +
==== [9] Jiménez-Fernández, S., Gurpegui, M., Díaz-Atienza, F., Pérez-Costillas, L., Gerstenberg, M., & Correll, C. U. (2015). Oxidative stress and antioxidant parameters in patients with major depressive disorder compared to healthy controls before and after antidepressant treatment: results from a meta-analysis. The Journal of clinical psychiatry, 76(12), 1658–1667. https://doi.org/10.4088/JCP.14r09179 ====
 +
 
 +
==== [10] Li, J., Yang, Z., Qiu, H., Wang, Y., Jian, L., Ji, J., & Li, K. (2020). Anxiety and depression among general population in China at the peak of the COVID-19 epidemic. World psychiatry : official journal of the World Psychiatric Association (WPA), 19(2), 249–250. https://doi.org/10.1002/wps.20758 ====
 +
 
 +
 
 +
==== [11] Vaváková, M., Ďuračková, Z., & Trebatická, J. (2015, May 20). Markers of Oxidative Stress and Neuroprogression in Depression Disorder. Oxidative Medicine and Cellular Longevity. https://doi.org/10.1155/2015/898393 ====
 +
 
 +
==== [12] Wang Y;Li H;Li T;He H;Du X;Zhang X;Kong J; (n.d.). Cytoprotective effect of Streptococcus thermophilus against oxidative stress mediated by a novel peroxidase (EfeB). Journal of dairy science. DOI: 10.3168/jds.2018-14601 ====
 +
 
 +
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 05:40, 19 October 2021


pro-efeB

Promoter

Profile

Name: pro-efeB

Base Pairs: 2107 bp

Origin: Escherichia coli str. K-12 substr. MG1655

Properties: Gene technology for protecting patented bacterial strains

Usage and Biology

In modern society, more and more people suffer from mild or severe depression. It has been reported that malondialdehyde (MDA) level in the plasma of depressed patients is significantly increased. After receiving conventional antidepressant treatment, the patient's MDA level decreased to the same as that of healthy people. Therefore, researchers believe that oxidative stress may play an important role in the occurrence and development of depression, and the activity of antidepressant therapy may be mediated by improving oxidative stress/antioxidant function. We attempt to express an antioxidant enzyme system (peroxidase gene efeB) in the cell or on the surface of the engineered probiotic bacteria to inhibit the production of malondialdehyde (MDA) by human cells and prevent cell oxidation from causing health damage to the body. So as to prevent or alleviate the condition of depression. The oxidation rate of peroxisome increases in proportion to the increase of oxygen tension. Especially in the case of high oxygen concentrations, the oxidation reaction of peroxisomes dominates. This characteristic allows peroxisomes to protect cells from the toxic effects of high concentrations of oxygen. efeB reduces the oxidative stress in the cell by competing with mitochondria for oxygen, and reduces the MDA produced during the oxidation of lipids.

Figure 1. Action and function of efeB in MDA reduction...

Construct design

Figure 2. The structure of efeB vector...

BBa_K3983002

Name: pro-efeB

Base Pairs: 2107 bp

Origin: Escherichia coli str. K-12 substr. MG1655

Properties: Gene technology for protecting patented bacterial strains

Peroxidase EfeB, originally studied as a substrate of the E. coli "double arginine" translocation system, is the first recognized peroxidase for bacterial dye decolorization. Subsequent studies have shown that EfeB is a peroxidase with heme as a prosthetic group, and has a specific iron transport function.We use E. coli as the starting strain and construct an engineered strain of efeB to explore the effects of efeB on bacterial growth, malondialdehyde concentration, ROS synthesis and antioxidant pathways under different oxidative stress conditions.

Engineering Success

Production, purification, and Agarose gel analysis of recombinant efeB

In order to present the function of the part, the efeB gene was expressed in E. coli under the control of T7 promoter. Then the bacterial cells are collected and crushed, and the efeB enzyme solution is purified by further confirmation by the Agarose gel electrophoresis, which is found in the corresponding protein band of approximately 2.1kb (Figure 3).

Figure 3 The result of Agarose gel electrophoresis...

Bacteria Growth

Bacteria A contains plasmid A, pUC57-efeB, and the bacteria D contains plasmid D, pUC57. Cultivate them in H2O2 solutions of different concentrations.When the concentration of hydrogen peroxide is 0 mM, the relative biomass of bacteria A and bacteria D are the same. As the concentration of hydrogen peroxide increases to 1mM, the relative biomass of bacteria A increases while the relative biomass of bacteria D remains the same, which shows that without efeB, bacteria is unable to grow when ROS is present. As the concentration of hydrogen peroxide increases to 2mM, the relative biomass of bacteria A decreases by half while the relative biomass of bacteria D decreases to 0mM. This shows that bacteria with the efeB gene is able to survive when the level of ROS increases. As the concentration of hydrogen peroxide increases to 4mM, the relative biomass of both bacteria A and bacteria D are zero. This shows that when the level of ROS is too high, bacteria are unable to survive regardless of the presence of the efeB gene.

Table 1. relative biomass percents of bacteria A and D with different concentrations of H2O2 ...
Figure 4. Histogram of relative biomass of bacteria A and D against different concentrations of H2O2 ...

Bacteria A contains plasmid A, pUC57-efeB, and the bacteria D contains plasmid D, pUC57. When the concentration of hydrogen peroxide is 0 mM, the relative biomass of bacteria A and bacteria C are the same. As the concentration of hydrogen peroxide increases to 1mM, the relative biomass of bacteria A increases while the relative biomass of bacteria C remains the same, which shows that without efeB, bacteria is unable to grow when ROS is present. As the concentration of hydrogen peroxide increases to 2mM, the relative biomass of bacteria A decreases by half while the relative biomass of bacteria D decreases to 0mM. This shows that bacteria with the efeB gene is able to survive when the level of ROS increases. As the concentration of hydrogen peroxide increases to 4mM, the relative biomass of both bacteria A and bacteria D are zero. This shows that when the level of ROS is too high, bacteria are unable to survive regardless of the presence of the efeB gene.

Reference

[1] McCarter T. (2008). Depression overview. American health & drug benefits, 1(3), 44–51.

[2] Chand SP, Arif H. Depression. [Updated 2020 Nov 18]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK430847/

[3] Wang, J., Wu, X., Lai, W., Long, E., Zhang, X., Li, W., Zhu, Y., Chen, C., Zhong, X., Liu, Z., Wang, D., & Lin, H. (2017). Prevalence of depression and depressive symptoms among outpatients: a systematic review and meta-analysis. BMJ open, 7(8), e017173. https://doi.org/10.1136/bmjopen-2017-017173

[4] Bueno-Notivol, J., Gracia-García, P., Olaya, B., Lasheras, I., López-Antón, R., & Santabárbara, J. (2021, January 1). Prevalence of depression during the COVID-19 outbreak: A meta-analysis of community-based studies. International Journal of Clinical and Health Psychology. DOI: 10.1016/j.ijchp.2020.07.007.

[5] U.S. Department of Health and Human Services (HHS). (n.d.). Major Depression. National Institute of Mental Health. https://www.nimh.nih.gov/health/statistics/major-depression#part_155720.

[6] Ferguson, J. M. (2001, February). SSRI Antidepressant Medications: Adverse Effects and Tolerability. Primary care companion to the Journal of clinical psychiatry. Doi: 10.4088/pcc.v03n0105

[7] Jiménez-Fernández, S., Gurpegui, M., Dí¬az-Atienza, F., Pérez-Costillas, L., Gerstenberg, M., & Correll, C. U. (2021, March 1). Comparison of ODD vs Healthy Controls. Psychiatrist.com. dx.dot.org/10.4088/JPC.14r09179.

[8] Rowe, L. A., Degtyareva, N., & Doetsch, P. W. (2008). DNA damage-induced reactive oxygen species (ROS) stress response in Saccharomyces cerevisiae. Free radical biology & medicine, 45(8), 1167–1177. https://doi.org/10.1016/j.freeradbiomed.2008.07.018

[9] Jiménez-Fernández, S., Gurpegui, M., Díaz-Atienza, F., Pérez-Costillas, L., Gerstenberg, M., & Correll, C. U. (2015). Oxidative stress and antioxidant parameters in patients with major depressive disorder compared to healthy controls before and after antidepressant treatment: results from a meta-analysis. The Journal of clinical psychiatry, 76(12), 1658–1667. https://doi.org/10.4088/JCP.14r09179

[10] Li, J., Yang, Z., Qiu, H., Wang, Y., Jian, L., Ji, J., & Li, K. (2020). Anxiety and depression among general population in China at the peak of the COVID-19 epidemic. World psychiatry : official journal of the World Psychiatric Association (WPA), 19(2), 249–250. https://doi.org/10.1002/wps.20758

[11] Vaváková, M., Ďuračková, Z., & Trebatická, J. (2015, May 20). Markers of Oxidative Stress and Neuroprogression in Depression Disorder. Oxidative Medicine and Cellular Longevity. https://doi.org/10.1155/2015/898393

[12] Wang Y;Li H;Li T;He H;Du X;Zhang X;Kong J; (n.d.). Cytoprotective effect of Streptococcus thermophilus against oxidative stress mediated by a novel peroxidase (EfeB). Journal of dairy science. DOI: 10.3168/jds.2018-14601

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 565
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 565
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 52
    Illegal BamHI site found at 284
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 565
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 565
    Illegal AgeI site found at 994
  • 1000
    COMPATIBLE WITH RFC[1000]