Composite

Part:BBa_K4165232

Designed by: Omnia AlaaEldeen Abdelazeem   Group: iGEM22_CU_Egypt   (2022-10-08)


(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.

Usage and Biology

The main function is the binding of the tau-binding peptide with the Cohasin protein. It has been used the 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

Modeling

For 2 Models with a 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 :

cbeta_deviations clashscore molprobity ramachandran_favored ramachandran_outliers Qmean_4 Qmean_6
1 2.74 1.22 97.11 0.52 1.96 1.88 


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

Comparison between the mathematical model of dry lab and the wey lab results of GST COH TD28 Rev using chemical lysis and sonicator

We have made a statistical analysis (t-test) to investigate the mathematical model's effectiveness in predicting the amount of the produced protein due to IPTG induction, whereas the test compares the mathematical model expected results to the replicates made in the induction experiment, given that all the condition of the experiment has been input to the model generating the predicted results. 

                Figure 3. This graph shows that the mathematical model of the dry lab and the wet lab results of GST COH 
                TD28Rev using chemical lysis doesn't correlate with each other as there is a great difference between the 
                two results, as the P-value is found to be <0.001.


               Figure 4. This graph shows that there is a correlation between the mathematical model of the dry lab and the 
                                          wet lab results of GST COH TD28Rev using sonicator



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