Difference between revisions of "Part:BBa K3983005"

 
 
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<partinfo>BBa_K3983005 short</partinfo>
 
<partinfo>BBa_K3983005 short</partinfo>
  
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pUC57-mini-amilGFP
+
promoter
 +
=== Profile ===
 +
 
 +
==== Name: amilGFP====
 +
==== Base Pairs: 699bp====
 +
==== Origin: Acropora millepora====
 +
==== Properties: A yellow chromoprotein====
 +
 
 +
=== 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...]]
 +
 
 +
=== Experimental approach ===
 +
 
 +
[[File:T--The Webb Schools--BBa K3983005-figure2.jpg|500px|thumb|center|Figure 2. Plasmid diagram...]]
 +
 
 +
=== BBa_K592010 ===
 +
 
 +
==== Name: amilGFP ====
 +
==== Base Pairs: 699bp ====
 +
==== Origin: Acropora millepora ====
 +
==== Properties: A yellow chromoprotein ====
 +
 
 +
In the process of cultivating microorganisms, namely Escherichia coli, because of their small size, they cannot be directly observed with the naked eye, and a certain method is needed for monitoring. In cell biology and molecular biology, the green fluorescent protein (GFP) gene is often used as a reporter gene. Through genetic engineering technology, the green fluorescent protein (GFP) gene can be transferred into the genomes of different species and continue to be expressed in offspring. . Therefore, we constructed the amilGFP engineered bacteria to show the growth status (number and vitality) of E. coli by observing the strength of the green fluorescent protein signal.
 +
 
 +
 
 +
=== Production, purification, and Agarose gel analysis of recombinant amilGFP ===
 +
In order to present the function of the part, the amilGFP gene was expressed in E. coli under the control of T7 promoter. Then the bacterial cells are collected and crushed, and the amilGFP  solution is purified by further confirmation by the Agarose gel electrophoresis, which is found in the corresponding protein band of approximately 700bp (Figure 3).
 +
 
 +
 
 +
[[File:T--The Webb Schools--BBa K3983005-figure3.jpg|500px|thumb|center|Figure 3...]]
 +
 
 +
=== Fluorescence and bacterial growth ===
 +
 
 +
We culture the engineered bacteria for 2hours under normal conditions, and then detect the intensity of the fluorescence value of the bacterial solution, and find that the fluorescence value is enhanced, indicating that E. coli grows normally.
 +
 
 +
[[File:T--The Webb Schools--BBa K3983005-figure4.png|500px|thumb|center|Figure4 The electrophoresis results of pUC57_mini_amilGFP enzyme digestion by BglI and HindIII. Lane M: KB Ladder. Lane 1:C845BGC060-6 plasmid.Lane 2:C845BGC060-6 plasmid digested by BglI and HindIII...]]
 +
 
 +
 
 +
[[File:T--The Webb Schools--BBa K3983005-figure5.jpg|500px|thumb|center|Figure5 Plate colonies under UV light...]]
 +
 
 +
 
 +
pUC57_mini plasmid and gene amilGFP were enzyme digestion. And then pUC57_mini backbone and amilGFP-fragment were connected by T4 ligase. Finally, we did double enzyme digestion by BglI and HindIII and sequenced for recombinant plasmid pUC57_mini_amilGFP. We got correct plasmid.
 +
 
 +
 
 +
=== 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 06:53, 19 October 2021


pUC57-mini-amilGFP

promoter

Profile

Name: amilGFP

Base Pairs: 699bp

Origin: Acropora millepora

Properties: A yellow chromoprotein

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...

Experimental approach

Figure 2. Plasmid diagram...

BBa_K592010

Name: amilGFP

Base Pairs: 699bp

Origin: Acropora millepora

Properties: A yellow chromoprotein

In the process of cultivating microorganisms, namely Escherichia coli, because of their small size, they cannot be directly observed with the naked eye, and a certain method is needed for monitoring. In cell biology and molecular biology, the green fluorescent protein (GFP) gene is often used as a reporter gene. Through genetic engineering technology, the green fluorescent protein (GFP) gene can be transferred into the genomes of different species and continue to be expressed in offspring. . Therefore, we constructed the amilGFP engineered bacteria to show the growth status (number and vitality) of E. coli by observing the strength of the green fluorescent protein signal.


Production, purification, and Agarose gel analysis of recombinant amilGFP

In order to present the function of the part, the amilGFP gene was expressed in E. coli under the control of T7 promoter. Then the bacterial cells are collected and crushed, and the amilGFP solution is purified by further confirmation by the Agarose gel electrophoresis, which is found in the corresponding protein band of approximately 700bp (Figure 3).


Figure 3...

Fluorescence and bacterial growth

We culture the engineered bacteria for 2hours under normal conditions, and then detect the intensity of the fluorescence value of the bacterial solution, and find that the fluorescence value is enhanced, indicating that E. coli grows normally.

Figure4 The electrophoresis results of pUC57_mini_amilGFP enzyme digestion by BglI and HindIII. Lane M: KB Ladder. Lane 1:C845BGC060-6 plasmid.Lane 2:C845BGC060-6 plasmid digested by BglI and HindIII...


Figure5 Plate colonies under UV light...


pUC57_mini plasmid and gene amilGFP were enzyme digestion. And then pUC57_mini backbone and amilGFP-fragment were connected by T4 ligase. Finally, we did double enzyme digestion by BglI and HindIII and sequenced for recombinant plasmid pUC57_mini_amilGFP. We got correct plasmid.


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
    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
    COMPATIBLE WITH RFC[1000]