Part:BBa_K4165255
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 689
Illegal SapI.rc site found at 85
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
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