Difference between revisions of "Part:BBa K4165013"
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                         variation of mRNA and protein concentrations with time compared with the wet lab results.
 
                         variation of mRNA and protein concentrations with time compared with the wet lab results.
 
===WetLab Results===
 
===WetLab Results===
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UBE2V2 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.
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UBE2V2 enzyme is a E2 ubiquitin-conjugating enzyme that has the ability to recruit the ubiquitin-protesome pathway, it was cloned in DH-5 alpha using pJET cloning vector and then expressed in BL21 using pGS-21a expression vector to be used in in-vitro ubiquitination assay to proof the concept that our system recruits the 26S protesomal-ubiquitin cascade.
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<p style=" font-weight: bold; font-size:14px;"> Transformation of His UBE2V2 in BL-21 using pGS-21a vector </p>
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<p style=" font-weight: bold; font-size:14px;"> Transformation of His UBE2V2 in BL-21 using pGS-21a expression vector and in DH-5 alpha using pJET cloning vector </p>
 
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/ube2v2-pgs.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/ube2v2-pgs.jpg" style="margin-left:200px;" alt="" width="500" /></p>
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Transformation was done using TSS protocol after testing different transformation protocols and optimizing the best conditions for the used protocol.
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Transformation was done using TSS protocol after testing three different buffers which are TSS buffer, Calcium Chloride and combination between Calcium Chloride and Magnesium Chloride. Transformation effeciency was calculated for Ube2V2 in pJET cloning vector and it was found to be 165000 No.of transformants/μg.
 
</html>
 
</html>
 
                                   Figure 2. Transformed plate of His UBE2v2 + pGS-21a  
 
                                   Figure 2. Transformed plate of His UBE2v2 + pGS-21a  
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/ube2v2-pjet.jpg" style="margin-left:200px;" alt="" width="500" /></p>
 
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/ube2v2-pjet.jpg" style="margin-left:200px;" alt="" width="500" /></p>
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Transformation was done using TSS protocol after testing different transformation protocols and optimizing the best conditions for the used protocol with a transformation efficiency = 165000 no. of transformants/ug and CFU/ml = 165000
 
 
</html>
 
</html>
 
                                     Figure 3. Transformed plate of His UBE2V2 + pJET  
 
                                     Figure 3. Transformed plate of His UBE2V2 + pJET  
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<p style=" font-weight: bold; font-size:14px;"> BCA of His UBE2V2 </p>
 
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<p><img src="https://static.igem.wiki/teams/4165/wiki/wet-lab/characterization/whatsapp-image-2022-10-12-at-5-22-44-pm.jpeg" style="margin-left:200px;" alt="" width="500" /></p>
 
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===

Revision as of 09:24, 13 October 2022


UBE2V2

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

Usage and Biology

This gene encodes a homolog of ubiquitin-conjugating enzyme E2 variant 1. These ubiquitin-conjugating enzymes don’t have the conserved cysteine residue critical for the catalytic activity of E2s. Based on the specific E2 used, the E2 enzymes can direct the ubiquitination process to different subsets of ubiquitin lysins. The formation of UBE2N/UBE2V2 complex facilitates the elongation of ubiquitination to forma a polyubiquitin chain. In our case, we will use its interaction with the UBE2N (E2 ligase) to catalyze the formation of polyubiquitin chains and the degradation of our targeted proteins.


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

UBE2V2 enzyme is a E2 ubiquitin-conjugating enzyme that has the ability to recruit the ubiquitin-protesome pathway, it was cloned in DH-5 alpha using pJET cloning vector and then expressed in BL21 using pGS-21a expression vector to be used in in-vitro ubiquitination assay to proof the concept that our system recruits the 26S protesomal-ubiquitin cascade.

Transformation of His UBE2V2 in BL-21 using pGS-21a expression vector and in DH-5 alpha using pJET cloning vector

Transformation was done using TSS protocol after testing three different buffers which are TSS buffer, Calcium Chloride and combination between Calcium Chloride and Magnesium Chloride. Transformation effeciency was calculated for Ube2V2 in pJET cloning vector and it was found to be 165000 No.of transformants/μg. 

                                  Figure 2. Transformed plate of His UBE2v2 + pGS-21a

Transformation of His UBE2V2 in DH-5 alpha 

                                    Figure 3. Transformed plate of His UBE2V2 + pJET

Refrences

1. UBE2V2 ubiquitin conjugating enzyme E2 v2 [homo sapiens (human)] - gene - NCBI. (n.d.). Retrieved September, from https://www.ncbi.nlm.nih.gov/gene/7336

2. David, Y., Ziv, T., Admon, A., & Navon, A. (2010). The E2 ubiquitin-conjugating enzymes direct polyubiquitination to preferred lysines. Journal of Biological Chemistry, 285(12), 8595-8604.

3. Pharos: Ubiquitin-conjugating enzyme E2 N (Tchem). (2022).

4. 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.