Composite

Part:BBa_K4165169

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


Biobrick His - CoH2

This composite part consists of T7 promoter (BBa_K3633015), lac operator (BBa_K4165062), RBS 4 (BBa_K4165264), 6x His-tag (BBa_K4165020), CoH2 (BBa_K4165003) and T7 terminator (BBa_K731721).

Usage and Biology

COH protein is a part of the Snitch system, which binds to the His 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 539
  • 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

Modeling

Model “peptide (His_Coh2) modeled by TRrosetta model1” Ranked the second model from our 4 ones with a score 5 out of 6 and values of:

cbeta_deviations clashscore molprobity ramachandran_favored ramachandran_outliers Qmean_4 Qmean_6
0 3.23 1.59 92.31 1.4 0.835 0.95 


                              Figure 1. The 3D structure of His-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.

Wet-Lab Results

A comparison between the Dry-Lab model and the Wet-Lab results of His-CoH2 part 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 the correlation between the mathematical model of the dry lab and the wet lab 
                                              results of His COH using sonicator. 
                    


             Figure 4. This graph shows that the mathematical model of the dry lab and the wet lab results of His 
                 COH using chemical lysis don't correlate with each other as there is a significant difference 
                 between the results, as the P-value is found to be <0.001


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Parameters
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