Difference between revisions of "Part:BBa K4165015"
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<p style=" font-weight: bold; font-size:14px;"> BCA of His UBE 2w </p>
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===Functional Parameters===
 
===Functional Parameters===

Revision as of 15:39, 12 October 2022


UBE2W

Ubiquitin-conjugating E2 ligase that has a role in the ubiquitination cascade for protein degradation.

Usage and Biology

This E2 ubiquitin-conjugating enzyme UBE2W is the key participant in the set of enzymes as it is responsible for the initial step of monoubiquitylation by TRIM21 E3 ligase. The UBE2W is most specific for RING domain E3 ligases which happens to be that Trim21, which we are working with, is one of those RING domain E3 ligases.


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
    COMPATIBLE WITH RFC[1000]

Dry Lab

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

UBE2W enzyme is a E2 ubiquitin-conjugating enzyme that has the ability to recruit the ubiquitin-protesome pathway, it was expressed in BL21 (DE3) to be used in in-vitro ubiquitination assay to proof the concept that our system recruits the 26S protesomal-ubiquitin cascade. UBE2W was characterized using BCA assay.

Transformation of His UBE 2w in BL-21 using pGS-21a vector

Transformation was done using TSS protocol after testing different transformation protocols and optimizing the best conditions for the used protocol with a transformation efficiency = 576000 no. of transformants/ug and CFU/ml = 1152000 

                               Figure 2. Transformed plate of His UBE 2w + pGS-21a

Transformation of His UBE 2w in DH-5 alpha using pJET vector

Transformation was done using TSS protocol after testing different transformation protocols and optimizing the best conditions for the used protocol with a transformation efficiency = 240000 no. of transformants/ug and CFU/ml = 480000 

                                 Figure 3. Transformed plate of His UBE 2w + pJET

BCA of His UBE 2w

<img src="whatsapp-image-2022-10-12-at-5-22-43-pm.jpeg"style="margin-left:200px;"alt=""width="500"/>

References

1. Stewart, M. D., Ritterhoff, T., Klevit, R. E., & Brzovic, P. S. (2016). E2 enzymes: more than just middle men. Cell research, 26(4), 423-440. 2. Vittal, V., Wenzel, D. M., Brzovic, P. S., & Klevit, R. E. (2013). Biochemical and structural characterization of the ubiquitin-conjugating enzyme UBE2W reveals the formation of a noncovalent homodimer. Cell biochemistry and biophysics, 67(1), 103-110.