Part:BBa_K4165173

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).

Source

Synthesized

Usage and Biology

The protac in our snitch system, it is responsible for connecting the target protein (tau) to the tTrim21 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, after ubiquitination the tau will be degraded by 26S proteosome.

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 : score 5 out of 6 , C-Beta deviation = 1 , clash score = 3.08 , molprobitey = 1.35 , Ramachandran favored = 96.31, Ramachandran outlier = 0.79 , Q Mean_4 = -0.01 , Q_Mean_6 = -0.29.

                          Figure 1. The 3D structure of the COH-Linker-WWW 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

Transformation of GST COH WWW in BL-21 using pGS-21a vector

                                    Figure 2. Transformed plate of GST COH (L) WWW + pGS-21a

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

                                   Figure 3. Transformed plate of GST COH (L) WWW + pJET 

Comparison between chemical lysis and sonication for GST COH WWW

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