Difference between revisions of "Part:BBa K4165009"

 
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===WetLab Results===
 
===WetLab Results===
<p style=" font-weight: bold; font-size:14px;"> Transformation of His Tau in DH-5 alpha using pJET cloning vector </p>
+
The tau protein is a protein responsible for the stabilization of microtubules that are essential for the establishment of cell polarity, the development of cell processes, and intracellular signal transduction. In AD tau is hyper-phosphorylated and that results in a change in his confirmation and starts to aggregate. We used the 0N4R tau gene starting with cloning with the pJET vector inside DH5 alpha bacterial cells. then we extract the plasmid to restrict the tau gene and ligate it with pGS-21a to transform it into BL-21. Then, we started the expression of tau protein inside BL-21 using IPTG. We extract the tau using two different methods, physical and chemical methods. After lysis, we purify tau using Ni-NTA affinity chromatography. Then we begin to build a tau aggregate model by incubation with heparin. To test the binding affinity between tau protein and two tau binding peptides which are TD28rev and WWW using pull-down assay, and to characterize the interaction we used the BCA assay. 
 +
<p style=" font-weight: bold; font-size:14px;"> Ligation of His Tau with pJET cloning vector </p>
 +
We used T4 ligase to ligate His Tau with pJET cloning vector so, we incubated His Tau with pJET overnight at 15°C.
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/ligation-of-tau.png" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
 +
          Figure 2. This figure shows the ligation between His Tau and pJET, His-Tau part size is 1338 bp so the band
 +
          expected to appear between 1500 bp and 1000 bp as shown in the gel. And for pJET plasmid size is 2974 bp so the
 +
          band expected to appear before 3000 bp band. So, for ligation of these two parts (plasmid + part) will be 4312
 +
          bp above 4000 bp band.
 +
 
 +
<p style=" font-weight: bold; font-size:14px;"> Transformation of His Tau in DH-5 alpha using pJET cloning vector and in Bl-21a using pGS-21a expression vector </p>
 +
The transformation was performed using TSS buffer as it shows the best transformation efficiency compared to Calcium Chloride and a combination of Calcium Chloride with Magnesium Chloride. The transformation efficiency was calculated for both His Tau in the pJET cloning vector and in the pGS-21a expression vector and they were found to be 10000 no. of transformants/ug and 576000 No. of transformants/μg respectively.
 
<html>
 
<html>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/his-tau-pjet.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/his-tau-pjet.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
</html>
 
</html>
                                     Figure 2. Transformed plate of His Tau + pJET.
+
                                     Figure 3. Transformed plate of His Tau + pJET.
 +
<p style=" font-weight: bold; font-size:14px;"> Miniprep of pJET cloning vector containing His Tau </p>
 +
Miniprep is a technique used to extract the plasmid containing the gene that encodes for the protein so, we performed miniprep for pJET containing His Tau.
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/miniprep-of-his-tau.png" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
 +
            Figure 4. This figure shows the miniprep of pJET cloning vector containing His Tau, The band appears with
 +
            size (4312 bp (2974 for pJET + 1338 for His-Tau)) which indicates that the miniprep was done
  
 
<p style=" font-weight: bold; font-size:14px;"> Transformation of His Tau in BL-21 using pGS-21a expression vector </p>
 
<p style=" font-weight: bold; font-size:14px;"> Transformation of His Tau in BL-21 using pGS-21a expression vector </p>
Line 76: Line 95:
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/his-tau-pgs.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/his-tau-pgs.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
</html>
 
</html>
                                     Figure 3. Transformed plate of His Tau + pGS-21a.
+
                                     Figure 5. Transformed plate of His Tau + pGS-21a.
  
 
<p style=" font-weight: bold; font-size:14px;"> SDS PAGE of induced and non induced samples of His Tau </p>
 
<p style=" font-weight: bold; font-size:14px;"> SDS PAGE of induced and non induced samples of His Tau </p>
 +
SDS-PAGE depends on the molecular weight of the protein. So, we used SDS-PAGE for His Tau to check if our protein is found in its exact size and to compare the induced and non-induced samples.
 
<html>
 
<html>
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/sds-of-his-tau.jpg" style="margin-left:200px;" alt="" width="500" /></p>
+
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/induction-of-tau.png" style="margin-left:200px;" alt="" width="500" /></p>
 
</html>
 
</html>
             Figure 4. This figure shows the comparison between the induced and non-induced samples of His Tau,  
+
             Figure 6. This figure shows the comparison between the induced and non-induced samples of His Tau,  
             where well no.2 is the non-induced sample while well no.5 is the induced sample showing that our  
+
             showing that our protein is induced effectively owing to our right choice of IPTG, time interval, and  
            protein is induced effectively owing to our right choice of IPTG, time interval and concentration
+
            concentration.
<p style=" font-weight: bold; font-size:14px;"> Pull down assay of Tau aggregates against GST COH WWW and GST COH TD28Rev </p>
+
<p style=" font-weight: bold; font-size:14px;"> BCA assay results for His Tau </p>
 +
BCA assay is a technique that is performed to check the concentration of the protein and it depends on the color of the BCA working solution which is directly proportional to the concentration of protein
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/standard-curve.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/bca-tau.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
 +
        Figure 7. BCA assay results of His Tau to check our protein concentration which is found to be 0.327679138.
 +
<p style=" font-weight: bold; font-size:14px;"> Affinity chromatography of His Tau </p>
 +
Affinity chromatography is a technique used to purify the proteins, to get the purified protein without the cell lysate so, we performed Affinity chromatography for His Tau to get the Tau protein to characterize it before performing pull down assay.
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/standard-curve.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/affinity-of-his-tau.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
 +
        Figure 8. This figure shows the BCA assay results after the affinity chromatography, after comparison induced
 +
        and non-induced absorption results with the standard curve our protein concentration is around (0.24866 ±
 +
        0.104462)
 +
<p style=" font-weight: bold; font-size:14px;"> Pull-down assay of Tau aggregates against GST Coh WWW and GST Coh TD28Rev </p>
 +
Pull-down assay is a one-step technique used to check the protein-protein interaction. Pull-down was performed to check the binding between the Tau aggregates with GST Coh WWW and GST Coh TD28Rev. Then we used the BCA assay to characterize the result of the pull-down. The results of pull-down show that the interaction between Tau aggregates with GST Coh WWW is better than that of Tau aggregates with GST COH TD28Rev as the concentration of elution of Tau aggregates with GST COH WWW is more than that of Tau aggregates with GST COH TD28Rev
 
<html>
 
<html>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/pull-down-of-tau-against-www-and-td28rev.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/pull-down-of-tau-against-www-and-td28rev.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
</html>  
 
</html>  
            Figure 5. This graph shows the comparison of pull down assay between Tau aggregates with GST COH WWW and GST COH
+
            Figure 9. This graph shows the comparison of the pull-down assay between Tau aggregates with GST Coh WWW  
              TD28Rev, showing that the interaction between Tau aggregates with GST COH WWW is better than that of Tau
+
                                                      and GST Coh TD28Rev.
              aggrergates with GST COH TD28Rev as the concentration of elution of Tau aggregates with GST COH WWW is more
+
              than that of Tau aggregates with GST COH TD28Rev
+
 
+
 
+
 
+
  
 +
<p style=" font-weight: bold; font-size:14px;"> Statistical analysis of the GST Coh (L) WWW and the pulldown with the tau protein </p>
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/data-analysis/pull-down/coh-www-tau.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 +
</html>
  
 +
      Figure 10. Statical analysis of the GST Coh (L) WWWW and pull-down of the GST Coh (L) WWW and the tau proteins show
 +
            that the pull down was successful in the Tau binding with significant efficiency to the WWW peptide.
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===

Latest revision as of 22:30, 13 October 2022


Tau (0N4R)

This basic part encodes the human microtubule-associated tau protein isoform 0N4R.


Usage and Biology

Alzheimer's disease (AD), which is considered the most common neurodegenerative disease to cause dementia, is characterized by 2 main accumulations that are amyloid plaques from amyloid beta and NFTs aggregates resulting from hyperphosphorylated or abnormally phosphorylated tau protein accumulation. Our part Tau, microtubule-associated protein (MAP) is a phosphoprotein that is prevalently found in cytosol and neuron axons. It is determined to be significantly expressed in neurons of the central nervous system (CNS) and the ocular tissues. It has a crucial role both under normal physiological conditions and also in the pathology of Alzheimer's disease.

In the normal brain, it can stabilize the neuronal microtubules that are essential for the establishment of cell polarity, the development of cell processes, and intracellular signal transduction. Six molecular tau isoforms are coded by a single gene on chromosome 17 resulting from alternative splicing of tau pre-mRNA and characterized to be significantly hydrophilic, heat stable, and soluble. These six isoforms differ in their binding repeats either 3R taus or 4R taus microtubule-binding repeats and the extra 4R repeat comes from the second (R2) repeat found in 4R. Tau biological activity is affected by 2 main processes that are alternative splicing and phosphorylation. For tau's interaction with tubulin and the enhancement of microtubule assembly, normal brain tau appears to require 2-3 moles of phosphate per mole of the protein. However, tau is phosphorylated at Ser262 and Ser214 in AD, which causes tau to separate from microtubules.

In the AD brain, tau Hyperphosphorylation is considered the main cause of AD progression, it may alter the protein's shape and charge, which in turn causes the microtubule-binding domain to become exposed and allow tau to self-assemble and form oligomers characterized to be neurofibrillary tangle. According to several studies, the polymerized tau (neurofibrillary tangles) is inert since it does not bind to tubulin or encourage its assembly into microtubules.

Before the development of NFTs, all six forms of tau are self-assembled into paired helical filaments as a result of hyperphosphorylation at the C-terminus of tau (PHFs). The aggregated tau protein takes this shape, which impairs axonal transit and continuously promotes microtubule instability. AD patients have an aberrant or hyperphosphorylated tau protein concentration that is four times higher than that of normal controls. These misfolded tau proteins are also recognized as potential neurotoxins and lose their basic function of MT stability along with enhanced aggregation effects.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 43
    Illegal AgeI site found at 229
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 207
    Illegal BsaI site found at 1119
    Illegal SapI.rc site found at 393

Dry Lab Characterization

Modeling

The structure of Tau was modeled by several tools and the top model was retrieved from trRosetta ranking 5 out of 6 according to our quality assessment code.


cbeta_deviations molprobity ramachandran_favored ramachandran_outliers Qmean_4 Qmean_6
0 3 95.28 1.05 -1.52844 -2.316739



                              Figure 1. Predicted 3D structure of Tau(0N4R) modeled by trRosetta


WetLab Results

The tau protein is a protein responsible for the stabilization of microtubules that are essential for the establishment of cell polarity, the development of cell processes, and intracellular signal transduction. In AD tau is hyper-phosphorylated and that results in a change in his confirmation and starts to aggregate. We used the 0N4R tau gene starting with cloning with the pJET vector inside DH5 alpha bacterial cells. then we extract the plasmid to restrict the tau gene and ligate it with pGS-21a to transform it into BL-21. Then, we started the expression of tau protein inside BL-21 using IPTG. We extract the tau using two different methods, physical and chemical methods. After lysis, we purify tau using Ni-NTA affinity chromatography. Then we begin to build a tau aggregate model by incubation with heparin. To test the binding affinity between tau protein and two tau binding peptides which are TD28rev and WWW using pull-down assay, and to characterize the interaction we used the BCA assay.

Ligation of His Tau with pJET cloning vector

We used T4 ligase to ligate His Tau with pJET cloning vector so, we incubated His Tau with pJET overnight at 15°C.

         Figure 2. This figure shows the ligation between His Tau and pJET, His-Tau part size is 1338 bp so the band 
          expected to appear between 1500 bp and 1000 bp as shown in the gel. And for pJET plasmid size is 2974 bp so the 
          band expected to appear before 3000 bp band. So, for ligation of these two parts (plasmid + part) will be 4312 
          bp above 4000 bp band.

Transformation of His Tau in DH-5 alpha using pJET cloning vector and in Bl-21a using pGS-21a expression vector

The transformation was performed using TSS buffer as it shows the best transformation efficiency compared to Calcium Chloride and a combination of Calcium Chloride with Magnesium Chloride. The transformation efficiency was calculated for both His Tau in the pJET cloning vector and in the pGS-21a expression vector and they were found to be 10000 no. of transformants/ug and 576000 No. of transformants/μg respectively.

                                   Figure 3. Transformed plate of His Tau + pJET.

Miniprep of pJET cloning vector containing His Tau

Miniprep is a technique used to extract the plasmid containing the gene that encodes for the protein so, we performed miniprep for pJET containing His Tau.

           Figure 4. This figure shows the miniprep of pJET cloning vector containing His Tau, The band appears with 
            size (4312 bp (2974 for pJET + 1338 for His-Tau)) which indicates that the miniprep was done

Transformation of His Tau in BL-21 using pGS-21a expression vector

                                    Figure 5. Transformed plate of His Tau + pGS-21a.

SDS PAGE of induced and non induced samples of His Tau

SDS-PAGE depends on the molecular weight of the protein. So, we used SDS-PAGE for His Tau to check if our protein is found in its exact size and to compare the induced and non-induced samples.

            Figure 6. This figure shows the comparison between the induced and non-induced samples of His Tau, 
            showing that our protein is induced effectively owing to our right choice of IPTG, time interval, and 
            concentration.

BCA assay results for His Tau

BCA assay is a technique that is performed to check the concentration of the protein and it depends on the color of the BCA working solution which is directly proportional to the concentration of protein

       Figure 7. BCA assay results of His Tau to check our protein concentration which is found to be 0.327679138.

Affinity chromatography of His Tau

Affinity chromatography is a technique used to purify the proteins, to get the purified protein without the cell lysate so, we performed Affinity chromatography for His Tau to get the Tau protein to characterize it before performing pull down assay.

       Figure 8. This figure shows the BCA assay results after the affinity chromatography, after comparison induced 
        and non-induced absorption results with the standard curve our protein concentration is around (0.24866 ± 
        0.104462)

Pull-down assay of Tau aggregates against GST Coh WWW and GST Coh TD28Rev

Pull-down assay is a one-step technique used to check the protein-protein interaction. Pull-down was performed to check the binding between the Tau aggregates with GST Coh WWW and GST Coh TD28Rev. Then we used the BCA assay to characterize the result of the pull-down. The results of pull-down show that the interaction between Tau aggregates with GST Coh WWW is better than that of Tau aggregates with GST COH TD28Rev as the concentration of elution of Tau aggregates with GST COH WWW is more than that of Tau aggregates with GST COH TD28Rev

           Figure 9. This graph shows the comparison of the pull-down assay between Tau aggregates with GST Coh WWW 
                                                     and GST Coh TD28Rev.

Statistical analysis of the GST Coh (L) WWW and the pulldown with the tau protein

     Figure 10. Statical analysis of the GST Coh (L) WWWW and pull-down of the GST Coh (L) WWW and the tau proteins show 
            that the pull down was successful in the Tau binding with significant efficiency to the WWW peptide.

References

1 - Iqbal, K., Liu, F., Gong, C. and Grundke-Iqbal, I., 2010. Tau in Alzheimer Disease and Related Tauopathies. Current Alzheimer Research, 7(8), pp.656-664.

2- Muralidar, S., Ambi, S., Sekaran, S., Thirumalai, D. and Palaniappan, B., 2020. Role of tau protein in Alzheimer's disease: The prime pathological player. International Journal of Biological Macromolecules, 163, pp.1599-1617.

3- R. Sajjad, R. Arif, A.A. Shah, I. Manzoor, G. Mustafa Pathogenesis of Alzheimer’s disease: role of amyloid-β and hyperphosphorylated tau protein Indian J. Pharm. Sci., 80 (2018), pp. 581-591, 10.4172/pharmaceutical-sciences.1000397

4- Köpke, E., Tung, Y., Shaikh, S., Alonso, A., Iqbal, K. and Grundke-Iqbal, I., 1993. Microtubule-associated protein tau. Abnormal phosphorylation of a non-paired helical filament pool in Alzheimer disease. Journal of Biological Chemistry, 268(32), pp.24374-24384.

5- Iqbal, K., Gong, C. and Liu, F., 2013. Hyperphosphorylation-Induced Tau Oligomers. Frontiers in Neurology, 4.

6- Gong, C. and Iqbal, K., 2008. Hyperphosphorylation of Microtubule-Associated Protein Tau: A Promising Therapeutic Target for Alzheimer Disease. Current Medicinal Chemistry, 15(23), pp.2321-2328.

7- Alonso, A., Cohen, L., Corbo, C., Morozova, V., ElIdrissi, A., Phillips, G. and Kleiman, F., 2018. Hyperphosphorylation of Tau Associates With Changes in Its Function Beyond Microtubule Stability. Frontiers in Cellular Neuroscience, 12.

8- Pérez, M., Cuadros, R., Smith, M., Perry, G. and Avila, J., 2000. Phosphorylated, but not native, tau protein assembles following reaction with the lipid peroxidation product, 4-hydroxy-2-nonenal. FEBS Letters, 486(3), pp.270-274.

9- Moszczynski, A., Gohar, M., Volkening, K., Leystra-Lantz, C., Strong, W. and Strong, M., 2015. Thr175-phosphorylated tau induces pathologic fibril formation via GSK3β-mediated phosphorylation of Thr231 in vitro. Neurobiology of Aging, 36(3), pp.1590-1599.

10- Lee, H., Perry, G., Moreira, P., Garrett, M., Liu, Q., Zhu, X., Takeda, A., Nunomura, A. and Smith, M., 2005. Tau phosphorylation in Alzheimer's disease: pathogen or protector?. Trends in Molecular Medicine, 11(4), pp.164-169.

11- Chu, D. and Liu, F., 2018. Pathological Changes of Tau Related to Alzheimer’s Disease. ACS Chemical Neuroscience, 10(2), pp.931-944.

12- Lin, Y., Cheng, J., Liang, L., Ko, C., Lo, Y. and Lu, P., 2007. The binding and phosphorylation of Thr231 is critical for Tau’s hyperphosphorylation and functional regulation by glycogen synthase kinase 3β. Journal of Neurochemistry, 103(2), pp.802-813.

13J. Neddens, M. Temmel, S. Flunkert, B. Kerschbaumer, C. Hoeller, T. Loeffler, V. Niederkofler, G. Daum, J. Attems, B. Hutter-Paier

14- Neddens, J., Temmel, M., Flunkert, S., Kerschbaumer, B., Hoeller, C., Loeffler, T., Niederkofler, V., Daum, G., Attems, J. and Hutter-Paier, B., 2018. Phosphorylation of different tau sites during progression of Alzheimer’s disease. Acta Neuropathologica Communications, 6(1).

15- Zhao, H., Chang, R., Che, H., Wang, J., Yang, L., Fang, W., Xia, Y., Li, N., Ma, Q. and Wang, X., 2013. Hyperphosphorylation of tau protein by calpain regulation in retina of Alzheimer's disease transgenic mouse. Neuroscience Letters, 551, pp.12-16.

16- Iqbal, K., Zaidi, T., Wen, G., Grundke-Iqbal, I., Merz, P., Shaikh, S., Wisniewski, H., AlafuzofT, I. and Winblad, B., 1987. Defective brain microtubule assembly in Alzheimer??s disease. Alzheimer Disease & Associated Disorders, 1(3), pp.201-202.

17- Bancher, C., Brunner, C., Lassmann, H., Budka, H., Jellinger, K., Wiche, G., Seitelberger, F., Grundke-Iqbal, I., Iqbal, K. and Wisniewski, H., 1989. Accumulation of abnormally phosphorylated τ precedes the formation of neurofibrillary tangles in Alzheimer's disease. Brain Research, 477(1-2), pp.90-99.

18- Braak, H., Braak, E., Grundke-Iqbal, I., & Iqbal, K. (1986). Occurrence of neuropil threads in the senile human brain and in Alzheimer's disease: A third location of paired helical filaments outside of neurofibrillary tangles and neuritic plaques. Neuroscience Letters, 65(3), 351-355. doi: 10.1016/0304-3940(86)90288-0

19- Rosenmann, H., Blum, D., Kayed, R., & Ittner, L. (2012). Tau Protein: Function and Pathology. International Journal Of Alzheimer's Disease, 2012, 1-2. doi: 10.1155/2012/707482

20- Sierra, H., Cordova, M., Chen, C., & Rajadhyaksha, M. (2015). Confocal Imaging–Guided Laser Ablation of Basal Cell Carcinomas: An Ex Vivo Study. Journal Of Investigative Dermatology, 135(2), 612-615. doi: 10.1038/jid.2014.371

21- Miao, J., Shi, R., Li, L., Chen, F., Zhou, Y., & Tung, Y. et al. (2019). Pathological Tau From Alzheimer’s Brain Induces Site-Specific Hyperphosphorylation and SDS- and Reducing Agent-Resistant Aggregation of Tau in vivo. Frontiers In Aging Neuroscience, 11. doi: 10.3389/fnagi.2019.00034

22- DeTure, M., & Dickson, D. (2019). The neuropathological diagnosis of Alzheimer’s disease. Molecular Neurodegeneration, 14(1). doi: 10.1186/s13024-019-0333-5

23- Uddin, M., Ashraf, G., Mamun, A., & Mathew, B. (2020). Toxic tau: structural origins of tau aggregation in Alzheimer's disease. Neural Regeneration Research, 15(8), 1417. doi: 10.4103/1673-5374.274329