Difference between revisions of "Part:BBa K4165255"

Revision as of 13:37, 13 October 2022

GST-Coh2-G4S- TD28REV

This part consists of the Cohesin 2 module (BBa_K4165003) connected to the tau binding peptide TD28REV (BBa_K4165006) through a flexible linker of GGGGS (BBa_K4165068) repeated 3 times and tagged with a GST tag (BBa_K4165070).

Usage and Biology

The part is considered an integral part of the snitch system in which it directs the Trim21 (E3) ligase to the tau protein in order to force the degradation of tau proteins which causes Alzheimer's Diseases via the proteasomal degradation pathway.

Sequence and Features

Dry-Lab Characterization

Modelling

                  Figure 1. A 3D model showing the structure of GST-CoH2-Linker-TD28REV (Model 3 - TRrosetta)

WetLab Results

In the wet lab we started with cloning in the pJET vector followed by the expression in the pgs21a, then we performed two different kinds of lysis to extract the protein to find which lysis buffer will give better yield, and quantified the protein expression before and after induction using BCA assay, in the end, we tested the GST COH TD28Rev affinity by pull down against His Trim21 (L) DOC and Tau aggregates to check their interaction.

Ligation of GST Coh TD28Rev with pJET cloning vector

We used T4 ligase to ligate GST Coh TD28Rev with pJET cloning vector so, we incubated GST Coh TD28Rev with pJET overnight at 15°C.

           Figure 2. This figure shows the ligation reaction between GST Coh TD28Rev with pJET cloning vector

Transformation of GST COH (L) TD21 Rev in DH-5 alpha using pJET cloning vector

The transformation was done using TSS buffer protocol, after trying three buffers which are Calcium chloride, Magnesium chloride and a combination between Calcium chloride and Magnesium chloride, we optimized our protocol to use the TSS buffer protocol as it showed the best results with a transformation efficiency of GST COH TD28Rev in DH-5 alpha using pJET vector is8.0×〖10〗^4/μg while that of GST COH TD28Rev in BL-21 using pGS-21a vector is 1.66×〖10〗^2/μg, you can find the complete protocol in our wiki page

                               Figure 3. Transformed plate of GST COH (L) TD28 Rev + pJET 

Transformation of GST COH (L) TD28 Rev in BL-21 using pGS-21a expression vector

                             Figure 4. Transformed plate of GST COH (L) TD28 Rev + pGS-21a

Comparison between chemical lysis and sonication for GST COH TD28 Rev

Chemical lysis and sonication were done to check which of them gives better results in the protein extraction, and after comparing the results we optimized our protocol to use chemical lysis for GST COH TD28Rev

                      Figure 5. this graph shows a difference between chemical lysis and sonication for GST COH TD28 
                       Rev, after we had the results we optimized our protocol to use chemical lysis for GST COH (L) 
                                                                TD28Rev.

SDS PAGE of induced and non induced samples of GST COH TD28 Rev

SDS depends on the molecular weight of the protein, we performed SDS to GST COH TD28rev to check that it is found in its exact size and to compare between the induced and non induced samples

               Figure 6. This figure shows the comparison between the induced and non induced samples of GST COH 
               TD28Rev, where well no.2 is the induced sample of GST COH TD28 Rev while well no.6 is the non induced 
               sample of GST COH TD28Rev showing that our protein is induced effectively owing to our right choice of 
                      I                       PTG, time interval and concentration

Pull down assay His Trim (L) DOC against GST COH WWW and GST COH TD28Rev

Pull down assay is a technique performed to check the interactions between the proteins and to check if they bind properly, we performed pull down assay to check the binding between His Trim21 (L) DOC with GST COH TD28Rev and to check the binding between tau aggregaTES and GST COH TD28Rev

               Figure 7. This graph shows the comparison of pull down assay between His Trim21 (L) DOC with GST COH 
               WWW and GST COH TD28Rev showing that the interaction between His Trim21 (L) DOC and GST COH WWW is 
               better than that of His Trim21 (L) DOC with GAT COH TD28Rev as the concentration of elution of His 
                  Trim21 (L) DOC with GST COH WWW is more than that of His trim21 (L) DOC with GST COH TD28Rev

Pull down assay of Tau aggregates against GST COH WWW and GST COH TD28Rev

               Figure 8. This graph shows the comparison of pull down assay between Tau aggregates with GST COH WWW and 
               Tau aggregates with GST COH TD28Rev, showing 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, illustrating that 
                           WWW could be a better candidate for binding to the Tau aggregates

Pull down assay between TD28rev and tau

SDS was performed after the pull down assay to check the protein-protein interaction

               Figure 8. This figure illustrates that the binding between GST COH TD28REv binds with tau as there are two 
                                                        bands that appear in the gel

BCA of GST COH TD28REV

BCA assay is a technique that is performed to quantify the proteins, and it depends on the color of the BCA dye which is directly proportional with the quantity of the protein, we performed BCA for GST COH TD28REV to know its concentration and it is found to be 0.497569224

               Figure 9. This graph illustrates the results of BCA assay for GST COH showing that our protein 
                                      concentration is expected to be 0.497569224