Composite

Part:BBa_K4165171

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


Biobrick GST - CoH2

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

Usage and Biology

COH protein is a part of the Snitch system, which binds to the GST tag for the purification of the COH.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1172
  • 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

Model “peptide (GST_Coh2) modeled by Alphafold GST_Coh2_f66fb_unrelaxed_rank_3_model_2” Ranked first model from our 4 ones with score 5 out of 6 and values of

cbeta_deviations clashscore molprobity ramachandran_favored ramachandran_outliers Qmean_4 Qmean_6
0 13.59 1.64 98.31 0.56 0.643 0.914 



                             Figure 1. The 3D structure of GST-COH 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 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 the correlation between the mathematical model of the dry lab and the wet lab 
                                             results of GST COH using chemical lysis 


          Figure 4. This graph shows that the mathematical model of the dry lab and the wet lab results of GST COH using 
           sonicator don't correlate with each as there is a difference between the results as the P-value is 
           found to be <0.05


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Categories
Parameters
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