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                                         Figure 3. Transformed plate of GST COH + pJET  
 
                                         Figure 3. Transformed plate of GST COH + pJET  
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<p style=" font-weight: bold; font-size:14px;"> Comparison between chemical lysis and sonication for GST COH </p>
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Figure 4. This graph shows a highly significant difference between the chemical lysis and sonication for GST COH, after we had this result we optimized our protocol to use sonication for GST COH
 
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===Functional Parameters===
 
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Revision as of 14:15, 11 October 2022


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).

Source

Synthesized

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

Dry lab

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 C Beta deviation = 0 , clash score = 13.59 , molprobitey = 1.64 , Ramachandran favored = 98.31 , Ramachandran outlier = 0.56 , Q Mean_4 = 0.643, Q Mean_6 = 0.914 


                             Figure 1. The 3D structure of COH model  Visualized by 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 1. 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

Transfotmation of GST COH in BL-21 using pGS-21a vector 

                                          Figure 2. Transformed plate of GST COH + pGS-21a

Transformation of GST COH in DH-5 alpha using pJET vector 

                                        Figure 3. Transformed plate of GST COH + pJET

Comparison between chemical lysis and sonication for GST COH

Figure 4. This graph shows a highly significant difference between the chemical lysis and sonication for GST COH, after we had this result we optimized our protocol to use sonication for GST COH