Difference between revisions of "Part:BBa K4165255"

Revision as of 19:05, 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 Disease 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 DH5 alpha using the pJET vector followed by manual miniprep to extract the plasmid which contains the part to restrict it and ligate it with pGS-21a and transform them into BL-21 to allow protein expression. Then we performed two different kinds of lysis to extract the protein to find which lysis buffer will give the better yield and quantified the protein expression before and after induction using BCA assay. In the end, we tested the GST Coh (L) 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 (L) 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 the 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 (L) TD28Rev in DH-5 alpha using pJET vector is 80000 no. of transformant/μg while that of GST Coh (L) TD28Rev in BL-21 using pGS-21a vector is 166 no. of transformant/μg, you can find the complete protocol in our wiki page.

                               Figure 3. Transformed plate of GST Coh (L) TD28Rev + pJET.

Transformation of GST Coh (L) TD28Rev in BL-21 using pGS-21a expression vector

                             Figure 4. Transformed plate of GST Coh (L) TD28Rev + pGS-21a

Miniprep of GST Coh (L) TD28Rev with pJET cloning vector

Miniprep is a technique used to extract the plasmid containing the gene that encodes for the protein so, we performed miniprep for pJET containing GST Coh (L) TD28Rev.

          Figure 5. This figure shows the miniprep of pJET containing GST Coh (L) TD28Rev, band appears with size 
              (4260 bp) (2974 for pJET + 1316 for Gst Coh2 (L) TD28) which indicates that the miniprep is done.

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 (L) TD28Rev. Then we used the BCA assay to check the best method for protein extraction. After the BCA results, we optimized our protocol to use the chemical method.

              Figure 6. This graph shows a difference between chemical lysis and sonication for GST Coh (L) TD28Rev.

SDS PAGE of induced and non-induced samples of GST Coh TD28Rev

SDS depends on the molecular weight of the protein, we performed SDS to GST Coh (L) TD28Rev to check that it is found in its exact size and to compare the induced and non-induced samples.

               Figure 7. This figure shows the comparison between the induced and non-induced samples of GST Coh (L) 
               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 (L) TD28Rev showing that our protein is induced effectively owing to our right choice of 
                      I                       IPTG, time interval, and concentration.

Pull-down assay His Trim (L) Doc against GST Coh (L) WWW and GST Coh (L) TD28Rev

Pull-down assay is a one-step technique performed to check the protein-protein interaction. To check if they bind properly, we performed the pull-down assay to check the binding between His Trim21 (L) DOC with GST Coh (L) TD28Rev and to check the binding between tau aggregates and GST Coh (L) TD28Rev.

               Figure 8. This graph shows the comparison of the pull-down assay between His Trim21 (L) Doc with GST Coh (L)
               WWW and GST Coh TD28Rev show that the interaction between His Trim21 (L) Doc and GST Coh (L) WWW is 
               better than that of His Trim21 (L) Doc with GST Coh (L) TD28Rev as the concentration of elution of His 
                  Trim21 (L) Doc with GST Coh (L) WWW is more than that of His trim21 (L) Doc with GST Coh (L) TD28Rev.

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

We tested the protein-protein interaction between tau aggregate and GST Coh (L) WWW and GST Coh (L) TD28Rev, then we characterize the pull-down using BCA assay to see the highest affinity between the 2 TBPs. The results show that the TPB WWW has high affinity than the TD28Rev.

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

SDS-PAGE for pull-down assay between TD28rev and tau

SDS-PAGE was performed after the pull-down assay to check the protein-protein interaction. If the proteins interact with each other the gel of SDS-PAGE will contain the 2 bands of the 2 proteins.

               Figure 9. This figure illustrates that the binding between GST Coh (L) TD28REv binds with tau as there are two 
                                                    bands that appear in the gel.

BCA of GST Coh (L) TD28Rev

BCA assay is a technique that is performed to quantify the proteins, and it depends on the color of the BCA working reagent which is directly proportional to the quantity of the protein, we performed BCA for GST Coh (L) TD28Rev to know its concentration and it is found to be 0.497569224.

              Figure 10. This graph illustrates the results of the BCA assay for GST Coh (L) TD28Rev showing that our 
                                     protein concentration is expected to be 0.497569224