Part:BBa_K4165256
GST-Coh2-G4S-WWW
This composite part encodes for Cohesin 2 module (BBa_K4165003) which is connected to the tau binding peptide WWW (BBa_K4165007) with 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 responsible for connecting the target protein (tau) to the Trim21 which binds to E2 conjugating enzymes and put tau and ubiquitin in proximity to each other resulting in ubiquitination of the tau proteins which causes Alzheimer's Diseases at lysine residue and after ubiquitination the tau will be degraded by 26S proteasome.
The protac in our snitch system is 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 SapI.rc site found at 85
Dry Lab characterization
We exhibited a pipeline to construct this part which runs as follows: modeling the fusion protein using different algorithms (ab initio and template-based tools for modeling), after that, we constructed a code to rank the 3D models from their json files. Upon choosing the best model, we docked the fusion protein with the tau protein to predict the binding affinity by which WWW peptide binds with the TAU protein, For more information please check our Docking page.
Modeling
From 3 models out of 16 Models modeled in 4 software, 1 Model ranked best according to its Q_Mean values 4 and 6. The best Model has values of :| cbeta_deviations | clashscore | molprobity | ramachandran_favored | ramachandran_outliers | Qmean_4 | Qmean_6 |
|---|---|---|---|---|---|---|
| 1 | 3.08 | 1.35 | 96.31 | 0.79 | -0.01 | -0.29 |
Figure 1. The 3D structure of the COH-Linker-WWW model Displayed on Pymol.
Mathematical modeling
Transcription rate and translation rate under T7 promotor
the mathematical modeling was based on our code for the calculation of transcription and translation (you can find it in the code section) beside with the estimated results from the wet lab.
Figure 2. this figure shows the results from the transcription and translation code showing the
variation of mRNA and protein concentrations with time compared with the wet lab results.
WetLab Results
In the wet lab, we started with cloning in the pJET vector followed by the expression in the pGS-21a. Then we performed two different kinds of lysis chemical and physical to extract the protein to find which lysis buffer will give a better yield, and quantified the protein expression before and after induction using BCA assay. In the end, we tested the GST Coh (L) WWW affinity by the pull-down assay against the His Trim21 (L) Doc and against the tau aggregates which showed that WWW could bind to the tau aggregates effectively.
Ligation of GST Coh (L) WWW with pJET cloning vector
We used T4 ligase to ligate GST Coh (L) WWW with pJET cloning vector so, we incubated Gst Coh (L) WWW with pJET overnight at 15°C.
Figure 3. This figure shows the ligation between GST Coh (L) WWW with pJET cloning vector, Our band is expected to be
at 4284 (A faint band appears). The ligation reaction didn’t ligate all the inserts so we will find two bands
of the vector and the insert at 2974 and 1310 respectively
Transformation of GST Coh (L) WWW in BL-21 using pGS-21a 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) WWW in DH-5 alpha using pJET vector is 14200000 transformants/µg while that of GST Coh (L) WWW in BL-21 using pGS-21a vector is 700 transformants/µg, you can find the complete protocol on our wiki page.
Transformation of GST Coh (L) WWW in DH-5 alpha using pJET vector
Figure 4. Transformed plate of GST Coh (L) WWW + pJET.
Miniprep for GST Coh (L) WWW in pJET cloning vector
Miniprep is a technique used to extract the plasmid containing our gene. So, we performed miniprep to extract the pJET cloning vector containing our GST Coh (L) WWW.
Figure 5. This figure shows the miniprep of pJET containing the GST Coh (L) WWW, Our mini prep band appears at
4284 (A faint band appears).
Figure 6. Transformed plate of GST Coh (L) WWW + pGS-21a.
Comparison between chemical lysis and sonication for GST COH WWW
Chemical lysis and physical lysis using 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) WWW.
Figure 7. This graph shows a significant difference between chemical lysis and sonication for GST Coh (L) WWW.
SDS PAGE for induced and non induced samples of GST COH WWW
SDS PAGE depends on the molecular weight of the protein. We performed SDS-PAGE to make sure that our protein is in the exact size and to show the difference between induced and non-induced samples of GST COH WWW.
Figure 8. This figure shows the comparison between induced and non-induced samples of GST Coh (L) WWW, where well no.1 is
the induced sample while well no.5 is the non-induced sample showing that our protein is induced
effectively owing to our right choice of IPTG, time interval, and concentration.
BCA assay results of GST Coh (L) WWW
BCA assay is a one-step 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) WWW to know its concentration and its is found to be 0.719466667.
Figure 9. This graph illustrates the results of BCA assay for GST Coh (L) WWW showing that our protein
concentration is expected to be 0.719466667 mg/ml.
Pull-down assay of His Trim21 (L) Doc against GST Coh (L) WWW and GST Coh (L) TD28Rev
Pull-down assay is a technique performed to check the protein-protein interaction and to check if they bind properly, we performed pull-down assay to check the binding between GST Coh (L) WWW against His Trim21 (L) Doc, and between Tau aggregates and GST Coh (L) WWW, where WWW was found to be a proper candidate of Tau (Illustrated in figure 5 and 6)
Figure 10. This graph shows the comparison of the pull-down assay between His trim21 (L) DOC with GST Coh (L) WWW and
GST Coh (L) TD28Rev, showing that the interaction between His Trim21 (L) DOC and GST COH WWW is better than
His Trim21 (L) DOC and GST COH TD28Rev as the concentration of the 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
Figure 11. This graph shows the comparison of pull-down assay between Tau aggregates with GST Coh (L) WWW and
GST Coh (L) TD28Rev, showing that the interaction between Tau aggregates with GST Coh (L) WWW is better than that
of Tau aggregates with GST Coh (L) TD28Rev as the concentration of elution of Tau aggregates with GST Coh (L) WWW
is more than that of Tau aggregates with GST Coh (L) TD28Rev, illustrating that WWW could be a better candidate for
binding to the Tau aggregates
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