Composite

Part:BBa_K4165173

Designed by: Ahmed Sameh   Group: iGEM22_CU_Egypt   (2022-09-30)


Biobrick GST - CoH2 - Linker - WWW

This composite part consists of T7 promoter (BBa_K3633015), lac operator (BBa_K4165062), RBS 4 (BBa_K4165264), GST (BBa_K4165070), CoH2 (BBa_K4165003), GGGGSx3 (BBa_K4165068), WWW (BBa_K4165007), and T7 terminator (BBa_K731721).

Usage and Biology

The protac in our snitch system is 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 at lysine residue and after ubiquitination the tau will be degraded by 26S proteasome.

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

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

Comparison between the mathematical model of dry lab and the wet lab results of GST COH WWW using sonicator and chemical lysis

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 
                                       WWW using sonicator correlate with each other


            Figure 4. This Graph shows the mathematical model of the dry lab and the results of the wet lab of 
            GST COH WWW using chemical lysis don't correlate with each other as there is a great difference between 
            the results, as the P-value is found to be <0.0001





[edit]
Categories
Parameters
None