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                Figure 5. this graph shows a difference between chemical lysis and sonication for GST COH TD28 Rev
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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) TD28 Rev
  
  

Revision as of 14:36, 11 October 2022


(GST) COH2-linker(G4S)-TD28REV

This biobrick consists of T7 promotor (BBa_K3633015), RBS (BBa_K4165016), GST (BBa_K4165070), COH2 (BBa_K4165003), G4S linker (BBa_K4165068), TD28REV (BBa_K4165006), Terminator (BBa_K731721), The GST tag was attached to the (COH2-Linker-TD28REV) coding sequence to serve in the purification using Glutathione resin column.

Source

synthesized

Usage and Biology

The main function is the binding of the tau-binding peptide with the Cohasin protein. It has been used in this project to target and degrade both tau and Aβ proteins which are both considered the main causes of Alzheimer’s Disease pathogenesis.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 45
    Illegal SapI.rc site found at 189

Dry lab

Modeling

For 2 Models with has score of 5 out of 6 and among 11 Models, we choose our best-ranked model according to its score of 5 out of 6 Q Mean_4 value. Our model values : C Beta deviation = 1 , clash score = 2.74 , molprobity = 1.22 , Ramachandran favored = 97.11 , Ramachandran Outlier = 0.52 , Q Mean_4 = 1.96 , Q Mean_6 = 1.88 


                   Figure 1. The 3D structure of the COH-linker-TD28rev model visualized by 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

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

                                Figure 3. Transformed plate of GST COH (L) TD28 Rev + pGS-21a

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

                               Figure 4. Transformed plate of GST COH (L) TD28 Rev + pJET

Comparison between chemical lysis and sonication for GST COH TD28 Rev

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) TD28 Rev