Difference between revisions of "Part:BBa K4165013"
 
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===Usage and Biology===
 
===Usage and Biology===
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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 lysines. 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.
+
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.
  
 
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===<span class='h3bb'>Sequence and Features</span>===
 
===<span class='h3bb'>Sequence and Features</span>===
 
<partinfo>BBa_K4165013 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4165013 SequenceAndFeatures</partinfo>
  
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===Dry Lab===
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<p style=" font-weight: bold; font-size:14px;"> Mathematical modeling </p>
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<p style=" font-weight: bold; font-size:14px;">Transcription rate and translation rate under T7 promotor </p>
 +
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.
 +
 +
<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/dry-lab/mathematical-modeling/ube2v22.png" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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                    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===
 
===WetLab Results===
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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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UBE2V2 enzyme is an E2 ubiquitin-conjugating enzyme that has the ability to recruit the ubiquitin-proteasome pathway. In the wet lab, we cloned UBE2V2 in DH-5 alpha using a pJET cloning vector. Then we extract the plasmid and restrict the gene of UBE2V2 to be ligated and expressed in BL21 using pGS-21a expression vector to be used in in-vitro ubiquitination assay to prove the concept that our system recruits the 26S proteasomal-ubiquitin cascade.
 +
<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>
 +
The transformation was done using the TSS protocol after testing three different buffers which are TSS buffer, Calcium Chloride, and a combination between Calcium Chloride and Magnesium Chloride. Transformation efficiency was calculated for UBE2V2 in the pJET cloning vector and it was found to be 165000 No. of transformants/μg.
 
<html>
 
<html>
 
<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>
 
</html>
 
</html>
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                                   Figure 1. Transformed plate of His UBE2v2 + pGS-21a  
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                                   Figure 2. Transformed plate of His UBE2V2 + pGS-21a  
 
<p style=" font-weight: bold; font-size:14px;"> Transformation of His UBE2V2 in DH-5 alpha </p>
 
<p style=" font-weight: bold; font-size:14px;"> Transformation of His UBE2V2 in DH-5 alpha </p>
 
<html>
 
<html>
 
<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>
 
</html>
 
</html>
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                                  Figure 2. Transformed playe of His UBE2V2 + pJET  
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                                    Figure 3. Transformed plate of His UBE2V2 + pJET  
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<p style=" font-weight: bold; font-size:14px;"> Affinity chromatography results for Ube2V2 </p>
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Affinity chromatography is a technique used to purify the proteins to get the protein alone without the cell lysate so, we performed affinity chromatography for total protein extraction to get pure UBE2V2.
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<html>
 +
<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/standard-curve.jpg" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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<html>
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<p><img src="https://static.igem.wiki/teams/4165/wiki/parts-registry/wetlab-results/ube2v2.jpg" style="margin-left:200px;" alt="" width="500" /></p>
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</html>
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              Figure 4. This figure shows the BCA assay results of affinity chromatography that was done after the
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              protein extraction.
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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3. Pharos: Ubiquitin-conjugating enzyme E2 N (Tchem). (2022).
 
3. Pharos: Ubiquitin-conjugating enzyme E2 N (Tchem). (2022).
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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.

Latest revision as of 19:16, 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 an E2 ubiquitin-conjugating enzyme that has the ability to recruit the ubiquitin-proteasome pathway. In the wet lab, we cloned UBE2V2 in DH-5 alpha using a pJET cloning vector. Then we extract the plasmid and restrict the gene of UBE2V2 to be ligated and expressed in BL21 using pGS-21a expression vector to be used in in-vitro ubiquitination assay to prove the concept that our system recruits the 26S proteasomal-ubiquitin cascade.

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

The transformation was done using the TSS protocol after testing three different buffers which are TSS buffer, Calcium Chloride, and a combination between Calcium Chloride and Magnesium Chloride. Transformation efficiency was calculated for UBE2V2 in the 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

Affinity chromatography results for Ube2V2

Affinity chromatography is a technique used to purify the proteins to get the protein alone without the cell lysate so, we performed affinity chromatography for total protein extraction to get pure UBE2V2. 

             Figure 4. This figure shows the BCA assay results of affinity chromatography that was done after the 
              protein extraction.


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.