Composite

Part:BBa_K4165074

Designed by: Hossam Hatem   Group: iGEM22_CU_Egypt   (2022-09-30)


T7 promoter-6x His tag-TRIM21-DocS-T7 terminator

This part is composed of T7 promoter (BBa_K3633015), lac operator (BBa_K4165062), RBS (BBa_K4165075), 6x His tag (BBa_K4165020), TRIM21 (BBa_K4165001), (G4S)3 linker (BBa_K157013), DocS (BBa_K3396000), and T7 terminator (BBa_K731721).

Usage and Biology

this part from the snith system which consists of the Trim part binds with the Docharin protein by a linker, this part will be purified using His tag.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 311
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 268
  • 1000
    COMPATIBLE WITH RFC[1000]

Modeling

After our Quality assessment process 2 Trim -the linker DoC model ranked first out of 15 models that were: 1- “peptide (trim-(GGGGS)3-docs) modeled by trRosetta model1.pdb” that had had score 5 with:

cbeta_deviations clashscore molprobity ramachandran_favored ramachandran_outliers Qmean_4 Qmean_6
0 141.09 2.61 98.57 0 3.73 2.3 



                      Figure 1. The 3D structure of the Trim Linker Docs model Displayed on Pymol.


2- ”peptide (trim-(GGGGS)3-docs) modeled by trRosetta model3.pdb” with a score 5 out of 6,

cbeta_deviations clashscore molprobity ramachandran_favored ramachandran_outliers Qmean_4 Qmean_6
0 140.33 2.61 99.43 0 3.98 2.66 



                      Figure 2. The 3D structure of the Trim Linker Docs 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 3. 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 (1mM IPTG concentration).



                Figure 4. 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 (2mM IPTG concentration).

WetLab Results

Comparison between the mathematical model of dry lab and the wet lab results of His Trim21(L) Doc 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 5. This graph shows that the mathematical model of the dry lab and the wet lab results of His Trim21 (L) Doc 
       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


        Figure 6. This graph shows the correlation between the mathematical model of the dry lab and the wet lab results of 
                                                 His Trim21 (L) DOC using a sonicator


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